Cytochrome P450IA1 is a polycyclic aromatic hydrocarbon (PAH)-responsive monooxygenase enzyme with no known endogenous inducer or substrate. We investigated the effect of suspension on P450IA1 gene (CYP1A1) expression in cultured human keratinocytes without the addition of xenobiotics. To prohibit adhesion and trigger differentiation, human keratinocytes or dermal fibroblasts were suspended in medium made semisolid with methylcellulose. Following suspension, we observed dramatic increases (> 100-fold) in steady state P450IA1 mRNA in keratinocytes within 1 h; however, dermal fibroblasts were nonresponsive. This effect was not dependent on methylcellulose itself and could be achieved by suspension in medium alone or in Percoll solution. The induction of P450IA1 mRNA was independent of exogenous calcium or serum concentrations, agents commonly used to signal differentiation. Adherent keratinocytes overlaid with methylcellulose exhibited slight morphological changes accompanied by increased P450IA1 mRNA. The activity of the P450IA1 enzyme was found to parallel Northern analysis data. Changes in keratinocyte adhesion or shape also affect other PAH-responsive genes suggesting Ah receptor involvement. We report a novel mechanism for cell type-specific induction of CYP1A1 expression without the addition of xenobiotic inducers. These findings suggest a possible endogenous role for P450IA1 in stratified squamous epithelia.