Overdiagnosis of HPV infection in cervical biopsies results in increased health care costs and unnecessary surgical procedures. Stringent criteria for histological diagnosis of koilocytosis were evaluated, using molecular detection of HPV DNA (polymerase chain reaction and Southern blot hybridization) as gold standard. Colposcopic biopsy specimens from 511 patients were studied, including 76 with referral diagnoses of negative cervix and 241 with CIN 1 or koilocytosis. Referral diagnoses for low-grade lesions failed to distinguish between HPV-infected and uninfected patients. False-positive rate for prediction of HPV infection was 74.8%. Biopsy specimens reevaluated using stringent diagnostic criteria showed increasing prevalence of HPV infection among patients whose biopsy specimens showed negative (43.7%), minimal (52.4%), or definite (69.5%) features of koilocytosis (P = .001). Similarly, subjects infected with high viral load or oncogenic HPV infection were more likely to be identified (P = .004 and .04, respectively). Despite increased predictive value of stringent diagnostic criteria, significant number of patients diagnosed as having CIN 1/koilocytosis (34.0%) did not in fact have HPV infection. Because most low-grade lesions spontaneously regress, patients with histological diagnosis of CIN 1 or HPV infection should be observed for a period of several months before definitive ablative treatment is undertaken.