Sensitive isotopic in situ hybridization analysis of 34 paraffin-embedded penile squamous cell lesions for human papillomavirus (HPV) types 6, 11, 16, 18, and 31 revealed similar HPV detection rates, HPV-type specificity, and viral distribution patterns to those described in analagous cervical and vulvar lesions. Human papillomavirus was detected in six of six cases of condylomata acuminata (HPV type 6 [n=4], HPV type 11 [n=2]), in six of eight cases of squamous cell carcinoma in situ (HPV type 16 [n=5], HPV type 31 [n=1]), and in four of 10 cases of squamous cell carcinoma (all were HPV type 16). Interestingly, all 10 cases of penile verrucous carcinoma analyzed were HPV negative. Reevaluation of the HPV-positive penile lesions with two commercial nonisotopic HPV-typing in situ hybridization kits (Pathogene, Enzo, New York, NY; Viratype, Life Technologies, Gaithersburg, Md), revealed positive results for HPV type 6 and/or type 11 in all six cases of condylomata acuminata studied. However, only the Viratype assay detected HPV genome in the high-grade squamous cell lesions, with a relative sensitivity of 70% compared with that of the isotopic assay.