Five HPV-33-immortalized and 5 HPV-33 + ras-transfected cell lines were characterized in terms of growth in soft agar, tumorigenic potential in nude mice, p21 expression, morphology and expression of differentiation markers in organotypic cultures. No striking differences were observed between the HPV-33-immortalized cell lines and their corresponding ras-transfected counterparts as regards their tumorigenicity in nude mice (only one cell line was able to develop tumors in nude mice) or their behavior on lifted collagen gels. However, all the ras-transfected cell lines gave rise to colonies in soft agar while only 2 HPV-33-transfected lines (CK1 and CK4) displayed this property. The 10 cell lines could be divided into 2 groups with respect to their phenotype in monolayer and in organotypic cultures. Lines from group I (CK2, 3, 5 and their ras-transfected homologous lines) shared a typical epithelial phenotype in monolayer and the ability (a) to form an epithelium similar to a CIN-III lesion and (b) to strongly express keratins K1-K10 and involucrin in organotypic cultures. On the other hand, for the lines from group II (CK1, CK4, CK1EJ7 and CK4EJ5), there was a correlation between an elongated phenotype in monolayer and the property (a) to form a structure similar to a microinvasive carcinoma and (b) to express vimentin and keratins K8-K18. These cell lines, exhibiting various transformation-associated alterations, can be considered as an in vitro model representing various stages of HPV-33-associated cervical carcinogenesis.