Modulation of ornithine decarboxylase (ODC) gene expression by retinoids was analyzed in human keratinocyte cultures maintained in serum-free medium containing 0.15 mM Ca++. Cells were incubated with all-trans-retinoic acid, 13-cis-retinoic acid or arotinoid Ro15-0778 (10(-10) to 10(-5) M), total RNA was isolated, and mRNA transcripts for ODC were analyzed by Northern and slot blot hybridizations with a human ODC cDNA. Treatment of cells for 24 h resulted in a dose-dependent decrease in ODC mRNA levels, with an estimated IC50 of approximately 1 X 10(-8) M for all-trans- and 13-cis-retinoic acid, while Ro15-0778 was somewhat less effective (IC50 approximately 1-5 X 10(-7) M). The suppression of ODC mRNA levels by retinoids was detectable at approximately 3 h of incubation, with essentially a maximal inhibition at 12 h. Reduced ODC mRNA levels noted after 24 h of incubation with 5 X 10(-7) M all-trans-retinoic acid were accompanied by a reduction in ODC enzyme activity. To determine if all-trans-retinoic acid was regulating ODC gene expression directly, or if protein synthesis was required, ODC expression was analyzed in cultures treated with protein synthesis inhibitors. In the presence of cycloheximide or puromycin, all-trans-retinoic acid did not suppress ODC mRNA levels. These findings suggest that suppression of ODC gene expression is not a direct effect of all-trans-retinoic acid, but depends on ongoing protein synthesis.