Numerous studies have demonstrated that killed suspensions of Corynebacterium parvum possess both lymphoreticular stimulatory and antitumor properties. In the present study, experiments were designed to isolate and characterize the component from C. parvum responsible for inducing stimulation of the lymphoreticular system and for inducing inhibition of progressive tumor growth. Fractions of C. parvum, prepared by hot phenol:water extraction of heat-killed bacteria, were tested in mice for lymphoreticular stimulation by measuring the degree of splenomegaly and spleen cell blastogenesis produced by an i.p. injection of the fractions. Antitumor activity was evaluated by measuring the extent of tumor growth after a s.c. injection of the C. parvum fractions admixed with either of three syngeneically transplanted murine tumors (B-16 melanoma, L1210 leukemia, or MC-93 sarcoma). Significant splenomegaly with concomitant increase in blastogenic activity was observed in mice treated with the residue after phenol:water extraction of C. parvum. The kinetics of development of splenomegaly and blastogenesis paralleled that observed by inoculation of whole organisms. In addition, significant antitumor activity, 80 to 100% inhibition of tumor growth, was observed in mice inoculated with an admixture of tumor cells and the extraction residue. The antitumor activity of the residue was similar to that observed with whole cells. The extracts (aqueous and phenol phases) did not induce splenomegaly, increased blastogenesis, or inhibition of tumor growth. Although purified cell walls had some antitumor activity (20 to 30% protection), the percentage of mice protected from tumor growth by cell walls was markedly less than that by whole organisms or the residue. Moreover, cell walls and/or protoplasm did not induce splenomegaly or increased blastogenesis. High antitumor- and splenomegaly-inducing activities of the residue were retained following chloroform:methanol extraction, sodium lauryl sulfate:pronase digestion, or sodium lauryl sulfate:pronase plus RNase:DNase:trypsin digestion, whereas these properties were significantly reduced after the residue was treated with metaperiodate, which resulted in a marked reduction in the carbohydrate concentration. Peritoneal cells from mice given an i.p. injection of whole organisms, phenol:water extraction residue, or periodate- or enzyme-treated residue were nonspecifically cytotoxic to B-16 tumor cells in vitro. However, tumoricidal activity of peritoneal cells did not develop in mice treated with either the aqueous or the phenol phase from extracted C. parvum. Collectively, results from this study demonstrate that the component in C. parvum responsible for antitumor activity and lymphoreticular stimulation is, in part, carbohydrate in nature and that, although nonspecific macrophage activation by whole cells or the extraction residue may be important, it is not sufficient for inducing tumor rejection. The possibility that full activity may depend upon the integrity of cell walls is discussed.