More than 42 types of human papillomaviruses are related to one another to varying degrees. Types 6, 11, 16, 18 and 31 are implicated in squamous carcinogenesis in the genital tract and oral cavity. Infection to types 6 and 11 usually lead to benign condyloma acuminata whereas Infections by types 16, 18 or 31 are often associated with high grade neoplasia, carcinoma in situ, and invasive carcinoma. Papillomaviruses can not be propagated in tissue culture systems. In vitro studies usually employ conventional biochemical methods on mass tissue specimens. Information on the distribution and quantity of viral DNAs and mRNAs, as well, as their relationship to cellular differentiation, or pathological status of the cells are all but lost. A few in situ studies on sectioned specimens using immunological or double- stranded DNA probes all indicated that the viral DNAs replicate and virus is packaged only in the upper, more differentiated epithelial cells. However, no mRNAs were ever detected in those studies. We propose to use single-stranded RNA probes with the in situ hybridization method of Angerer & Angerer to examine routinely processed biopsy specimens of different squamous neoplasms to study the biology of these viruses and their relationship to the diseases they cause. We have developed, for each of the five genital viruses, type specific probes that do not cross react even with closely related viruses in Southern blotting analyses. We will use in situ hybridization on the biopsies to test the utility of these probes in clinical diagnosis. We will also develop, for each of the five genital viruses, probes that are specific for mRNA species that we have identified through our other studies. We will establish the correlation between the various neoplasms with the presence of the different types of HPVs and examine HPV gene expression in individual cells in different grades of neoplasia. Our preliminary studies on 20 condyloma acuminata and one carcinoma show that mRNAs and DNAs are detected in routinely processed biopsies and mRNA signals are much higher than those of DNA. Eventually we wish to develop these probes to be routine, clinical diagnostic as well as research tools.