Genomic insertion of human papillomavirus (HPV) sequences is associated with the genesis of cervical carcinoma, and HPV-induced incipient cellular alterations may also present a requisite for the establishment of cell lines such as HeLa. Considering the theoretical importance of specific viral integration sites, we attempted to detect in HeLa cells the chromosomal location of DNA sequences homologous to HPV-16 and HPV-18 sequences by a nonisotopic high resolution in situ hybridization technique. Chromosome identification following in situ hybridization was possible by counterstaining of the same preparation with Chromomycin A3, Distamycin A, and DAPI. Using this approach, we have assigned HPV-18 integration in HeLa cells to band 8q24 (a site including the locus of the myc-protooncogene), to an abnormal chromosome 22, and to a not yet identified marker chromosome possibly neighboring other oncogenic or activating sites. The sensitive detection technique described in this study presents a new approach involving in situ chromosome hybridization with biotinylated DNA probes in combination with reflection contrast microscopy and subsequent fluorescent R- and C-banding. The method allowed the assignment of a 7-kb HPV-18 DNA probe to human chromosomal sites important in growth regulation and cancerogenesis. It should prove useful in a number of similar studies using other viral and oncogenic DNA probes.