The spontaneous human keratinocyte line HaCaT and c-Ha-ras oncogene-transfected cell clones are capable of expressing an unusually broad spectrum of keratins, not observed so far in epithelial cells. This expression is, however, strongly modulated by environmental conditions, including cell density. Both cells of the nontumorigenic HaCaT line and the tumorigenic HaCaT-ras clones, I-7 and II-3 (giving rise to benign and malignant tumors, respectively), constitutively expressed the keratins K5, K6, K14, K16 and K17, which are also common in cultures of normal keratinocytes. In addition keratins K7, K8, K18 and K19, generally associated with simple epithelia, were synthesized (to a most pronounced extent in sparse cultures), while keratins K4, K13 and K15 appeared at confluence, presumably with the onset of stratification. Moreover, in both HaCaT and HaCaT-ras clones the epidermal "suprabasal" keratins, K1 and K10, were expressed in conventional submerged cultures (at normal vitamin A levels), markedly rising with cell density, but not strictly correlated with the degree of stratification. This property was maintained in HaCaT cells up to the highest passages. According to immunofluorescence, this was due to increasing numbers of strongly stained cells, and not due to a gradual increase in all cells. Most strikingly, there was a significant delay in the appearance of K10 compared to K1, and this dissociation of expression was most evident in dispase-detached cell sheets (submerged cultures) and organotypic cultures of the ras clones (grown at the air-liquid interface). While on frozen sections bright staining for K1 was seen in some basal and virtually all suprabasal cell layers, K10 was largely restricted to the uppermost layers. Thus, obviously synthesis of K1 and K10 can be regulated independently, although generally in this given sequence. The apparent compatibility of K1 synthesis with proliferation and particularly the extended delay of K10 expression (as a postmitotic event) might be causally related to altered growth control and as such imply the significance of this disturbance. Finally, the highly preserved epidermal characteristics, in terms of expression of keratins (and other differentiation markers [5]) and their regulation, makes these cell lines excellent candidates for studying external modulators of differentiation and also underlying molecular mechanisms.