Ten immortalized cell lines were established by transfection of human cervical keratinocytes (CK) with HPV-33 DNA and some of their characteristics were investigated. The following observations were made: (a) several cell lines have reached over 100 population doublings in vitro; (b) 3 transcripts were observed, 2 being encoded by the E6/E7 open reading frames (ORFs); (c) cytogenetic analyses showed important genetic modifications such as aneuploidy and isochromosome formation of the q arm of chromosome 8; (d) 2 of the 10 cell lines developed colonies in soft agar but none was able to form tumors when injected s.c. into nude mice; (e) Southern analysis suggested that a single copy of HPV-33 is integrated at a single common site within the genome of the 10 cell lines. These immortalized cell lines should be useful for studying mechanisms involved in proliferation, differentiation and neoplastic transformation of CK by HPV-33.