Dictyostelium discoideum is a simple eukaryote that lives as an amoeba until starvation triggers aggregation. The aggregate forms a slug which then develops into a fruiting body with two main cell types, stalk and spore cells. Two mechanisms have been proposed to explain cell-type differentiation. Studies using expression of the ecmA gene as a prestalk cell marker indicated that gradients of morphogens determine cell fate in the slug. However, studies using dyes or the cysteine proteinase 2 (CP2) gene product as a prestalk cell marker indicated that cell autonomous factors such as cell-cycle phase at the time of starvation cause an initial choice of cell fate. To help resolve these differences, we have used transformed cells containing the promoter of the prestalk gene ecmA fused to beta-galactosidase (Jermyn and Williams, 1991) to study the differentiation of Dictyostelium cells at low cell density, at which cell-to-cell interactions and morphogen gradients are minimal. We find that under all conditions of low cell density in which express ion the ecmA fusion gene occurs, it is invariably detected in less than 25% of the cells from a clonal population. This suggests that a cell-autonomous mechanism is involved in ecmA expression. We then used double-labeled immunofluorescence to examine the ontogeny of the CP2-positive and the ecmA-positive cells. In developing aggregates, 9 to 12% of the cells are CP2-positive from 12 to 24 hr of development. The ecmA-positive cells are first detected at 16 hr as a subset of the CP2-positive cells and then increase in number. At approximately 20 hr, the CP2-positive cells and the ecmA-positive cells are almost completely overlapping sets. By late development, all of the CP2-positive cells are ecmA-positive and an additional 10% of the CP2-negative cells are also ecmA-positive. This indicates that up to 20 hr development, ecmA is expressed only in CP2-positive cells. The data thus suggest that cell-cycle phase at the time of starvation causes an initial choice of cell type and that during later development other factors influence cell fate.