Retinoids present in cultured human epidermal keratinocytes maintained in medium containing 25 nM retinol were identified and quantitated utilizing reverse-phase gradient high performance liquid chromatography. Total cell retinoid mass and composition averaged 12.59 pmol/mg cell protein +/- 1.86, (mean +/- S.D., n = 5) between strains and was constant with culture age. Long chain fatty acid esters of retinol and 3,4-didehydroretinol comprised greater than 95% of total retinoid, each contributing 77 and 23% of total esters, respectively. In mature stratified cultures, less abundant retinoids included unesterified retinol, 1%, and 3,4-didehydroretinol, 0.5%, retinoic acid, 0.5%, and a retinoid not previously reported in mammalian cells, 3,4-didehydroretinoic acid, 0.3%. This absolute and relative retinoid composition was essentially constant with culture age except that 3,4-didehydroretinoic acid was not detected in 3-4-day-old cultures (16-32 cell colonies), suggesting that keratinocyte differentiation may be related to endogenous changes in production of “active” retinoids. Separate pools of retinyl and 3,4-didehydroretinyl esters served as predominant sources of substrate for production of retinoic and 3,4-didehydroretinoic acids, respectively, suggesting that their production may be regulated independently.