Regulation of ornithine decarboxylase (ODC) gene expression and cell growth by all-trans-retinoic acid in the presence and absence of exogenous putrescine were examined in normal keratinocyte cultures maintained in serum-free medium containing 0.15 mM Ca++. Putrescine and the higher polyamines are negative feedback regulators of ODC synthesis and are essential for cell growth. Human keratinocytes were incubated with and without 1 microM putrescine and the effects of 5 x 10(-7) M retinoic acid on ODC mRNA levels, ODC activity, polyamine levels, and DNA synthetic rates were determined. Northern blot analysis of total RNA isolated from breast reduction keratinocytes treated with retinoic acid up to 24 h showed a time-dependent suppression of ODC mRNA levels that was unaffected by putrescine. ODC activity was suppressed more rapidly in keratinocytes grown in the absence of putrescine; however, at 24 h, ODC activity was suppressed to an equal extent under both culture conditions. The effect of retinoic acid on polyamine levels was determined in the absence of exogenous putrescine. Retinoic acid treatment markedly suppressed putrescine and N1-acetylspermidine levels, whereas spermidine and spermine levels were relatively unaffected. The effect of retinoic acid on DNA synthetic rates, as measured by 3H-thymidine incorporation, was variable. Retinoic acid either stimulated or had little effect on keratinocyte DNA synthetic rates in cells derived from breast reductions and cultured in the absence of putrescine; these effects were not opposed by the presence of exogenous putrescine. In contrast, DNA synthesis in keratinocytes derived from neonatal foreskins was consistently suppressed by retinoic acid, independent of the polyamine status. Our data, therefore, suggest that the effect of retinoic acid on cell growth, as indicated by DNA synthetic rates, does not necessarily parallel its effect on ODC activity and mRNA levels.