Retinoic acid (RA) modulates epidermal homeostasis and affects differentiation-associated proteins such as keratin K1 and filaggrin. Because results from in vitro and in vivo studies have been conflicting with respect to RA effects on keratinization, we examined the terminal differentiation of epidermal cell cohorts after RA stimulation in vivo. Pulse-labelling with 5-bromo-2-deoxyuridine (BrdU) was performed by intraperitoneal injection of mice immediately or at 16 h after a single topical application of 100 nmol RA. The cell cohort labelled at the time of RA application consisted of previously unperturbed cells exposed to RA after initiation of S-phase, whereas the cohort labelled 16 h after RA application consisted of cells stimulated into the S-phase by RA. These two cohorts of partially synchronized cells were followed for up to 72 h after BrdU labelling. Such labelling combined with keratin K1 or filaggrin expression was scored by paired immunofluorescence staining of skin sections. The onset of keratin K1 expression was unchanged in both series after RA treatment, while filaggrin appeared earlier than in controls. The differential effect of RA on the maturation markers was related to the proliferative activity, the increased cell turnover, and the shortened epidermal transit time. The onset of keratin expression appeared to be regulated before the postmitotic period, whereas filaggrin expression appeared to be regulated during the late phase of the maturation process, thus being influenced by the actual epidermal kinetics and structural alterations. These results suggested that the effect of RA on epidermal differentiation is secondary to its effect on proliferation, as determined by the altered cellular age distribution following regenerative proliferation.