A case-control study of 525 cases of cervical intraepithelial neoplasia grade III (CIN III) and 512 controls was conducted in Spain and Colombia between 1985 and 1988 to assess the role of human papillomavirus (HPV) in the etiology of CIN III. HPV DNA in cytological scrapes from the cervix was assessed by Virapap and by polymerase chain reaction (PCR) based on the L1 consensus primers. A subsample of 268 specimens was also tested for HPV DNA using Southern hybridization. In Spain, the PCR-based prevalences of HPV DNA were 70.7% among cases and 4.7% among controls. Odds ratio (OR) and 95% confidence interval (numbers in parentheses) for HPV DNA were 56.9 (24.8-130.6). In Columbia HPV DNA was detected by PCR in 63.2% of the cases and in 10.5% of the controls. The OR was 15.5 (8.2-29.4). The estimated fractions of CIN III attributable to HPV were 72.4% in Spain and 60.3% in Colombia. HPV 16 was the predominant viral type and showed the strongest association with CIN III; in Spain the OR was 295.5 (44.8-1946.4) and in Colombia the OR was 27.1 (10.6-69.5). HPV DNA of unknown type was frequent in HPV-positive cases (18.3% in Spain and 38.0% in Colombia) and controls (66.7% in Spain and 47.4% in Colombia). The comparison of results from Virapap and PCR indicated that PCR is the method of choice for epidemiological studies. These data strongly support the hypothesis of the viral origin of CIN III, the common etiology of CIN III and invasive cervical cancer, and the causal nature of the association between HPV and CIN III.