A number of epidemiological studies indicate that cigarette smokers are at increased risk of developing cervical cancer. However, convincing biological evidence is lacking. This report examines the biological and cellular role of human papillomavirus (HPV) type 16 and cigarette smoke in multistage cervical carcinogenesis. Two lines of HPV 16-immortalized human endocervical cells (HEN-16 and HEN-16-2) generated from primary cells (HEN) were treated with cigarette smoke condensate (CSC). CSC-treated, but not untreated, HEN-16 and HEN-16-2 formed tumors that were invasive squamous cell carcinomas in nude mice. The tumors were used to initiate 2 tumor lines of cells (HEN-16T and HEN-16-2T, respectively). Cells of both tumor lines, compared with HEN, HEN-16 and HEN-16-2, featured: (a) tumorigenicity, (b) distinct morphologies in monolayer and organotypic (raft) cultures, (c) faster growth in serum plus high calcium levels after immortalization and after transformation, (d) higher saturation density and (e) anchorage-independent growth. Our results provide unique direct in vitro evidence that cigarette smoke causes cancer in HPV-containing cervices.