The BrdU/Hoechst 33258/Giemsa method for sub-dividing S-phase in asynchronous cell populations has been re-evaluated and modified to give better definition and more even distribution of sub-phases. A reference pattern of early-replicating euchromatic bands is given for all chromosomes at Sk2 in primary cultures of skin fibroblasts. The overall band patterns at each sub-phase have allowed more objective definitions of "early" and "late" replication for these cells, and show that in both classes of chromatin light G-bands preceed dark G-bands. Asynchrony between homologous bands is observed at all stages of S, albeit with a variable frequency. The observed in vitro replication patterns and programme for the chromosomes of skin fibroblasts does not appear to be affected by the age or sex of the source.