Undifferentiated cervical carcinomas vary considerably in their intercellular organization and patterns of invasion. In spite of its clinical significance, the basis for such variation is poorly understood. We investigated the cellular properties that may be responsible for this diversity, using as a model two human cervical carcinoma cell lines that were derived from the same tumor specimen and the same clone. It was shown previously that, in spite of their common origin, each line forms a histologically distinct type of undifferentiated carcinoma when heterotransplanted in vivo: cells of line C-4I grow as compact expanding masses with central necrosis, while tumors of line C-4II infiltrate host tissues as small, well-vascularized, dispersed cell groups. The characteristic behavior of each line was retained in culture, where C-4I cells formed highly multilayered cohesive colonies, while C-4II cells formed diffuse, monolayered colonies and shed into the culture medium. These observations as well as ultrastructural data suggested that each line may be arrested at a different stage of stratified squamous differentiation. In the present study, this hypothesis was tested by examining specific differentiation markers. An analysis of the cultures by immunofluorescence microscopy and immunoblotting revealed that keratin was more abundant in the compact C-4I line than in the dispersed C-4II line. C-4I cells expressed keratins 5, 6, 8, 16, 18, and 19, while C-4II expressed only keratins 8, 16, 18, and 19. In the multilayered C-4I colonies, involucrin-positive cells occurred in the apical cell layers only. In C-4II, involucrin-positive cells occurred in monolayers and domes, and they were most consistently located apically in crowded cultures. Laminin was secreted by both lines, but only C-4II cells deposited a fibronectin matrix. The results suggest that C-4I cells resemble normal cervical cells at the spinous stage of stratified squamous differentiation, while C-4II cells resemble basal/suprabasal cells. The different growth patterns of the tumors, formed by the lines in vivo, therefore likely reflect functional and behavioral differences that normally exist between spinous and basal cervical epithelial cells. The results suggest that differentiation-related functional properties may lead to histological diversity among cervical carcinomas that are categorized as undifferentiated by histopathological criteria.