Secondary mitotic human skin fibroblast populations in vitro underwent 53 +/- 6 cumulative population doublings (CPD) in 302 +/- 27 days. When the growth capacity of the mitotic fibroblasts is exhausted, and if appropriate methods are applied, the fibroblasts differentiate spontaneously into postmitotic fibroblast populations, which were kept in stationary culture for up to 305 +/- 41 additional days. Mitotic and postmitotic fibroblast populations are heterogeneous populations with reproducible changes in the proportions of mitotic fibroblasts F I, F II, and F III, and postmitotic fibroblasts F IV, F V, F VI, and F VII. This process makes it evident that the fibroblasts differentiate spontaneously along a seven-stage terminal cell lineage F I-F II-F III-F IV-F V-F VI-F VII. Shifts in the frequencies of the mitotic and postmitotic fibroblasts in mass populations are accompanied by alterations in the [35S]methionine polypeptide pattern of the developing mass populations. The [35S]methionine polypeptide patterns of homogeneous subpopulations of F I, F II, F III, F IV, F V, and F VI isolated from heterogeneous mass populations reveal that the six fibroblast morphotypes studied express their cell-type-specific [35S]methionine polypeptide pattern in the heterogeneous mass populations.