The relationship between transformation, lactate production, and glucose transport was examined in a series of ten cell lines consisting of subclones of BALB 3T3 A31 cells and viral and chemical transformants of either the subclones or the original A31 line. Comparisons were made over a relatively narrow range of cell densities to minimize changes in the biochemical parameters during growth. A nitroquinoline oxide (NQT-3T3-714) and a temperature-sensitive Kirsten sarcoma virus (tsKi-3T3-714) transformant of subclone 714 exhibited transformed phenotypes with respect to morphology and growth properties, but their rates of lactate production and 2-[3H]deoxy-d-glucose (deoxyglucose) uptake were similar to those of the parent cells. 2- to 5-fold in these transformants, showing that there was no defect in the enzymes of this pathway. At a temperature nonpermissive for transformation of tsKi-3T3-714, lactate production by this line did not decrease relative to the rate of the parent cells. Another transformant, Ki-3T3-234, had a glycolytic rate which was 4 to 5 times greater than that of the low lactate producers while other transformants exhibited intermediate rates, and the rate of a third nontransformed 3T3 A31 subclone, K-1-1, was comparable to the rate of Ki-3T3-234. The rates of [3H]deoxyglucose uptake by this series of cells were closely proportional to their glycolytic rates rather than to their state of transformation. Increasing glycolysis by oligomycin or dinitrophenol treatment, however, did not cause a concomitant increase in sugar uptake. Neither glycolysis nor deoxyglucose uptake in the high-lactate producer (Ki-3T3-234) was inhibited by ouabain, suggesting that Na+-K+-adenosinetriphosphatase is not a regulator of these functions in 3T3 cells. In 3T3-derived cells, it appears that the rates of glycolysis and glucose uptake may be regulated in tandem under some conditions and that neither process is an obligatory consequence of neoplastic transformation.