Prespore and prestalk cells can be distinguished within aggregates of Dictyostelium by the expression of well-characterized cell type-specific genes. Fusion of the tagB regulatory region to Escherichia coli beta-galactosidase revealed that this prestalk specific gene marks the differentiation of the initial prestalk cell population, PST-1. The reporter gene was expressed normally in tagB- mutant cells despite the fact that they do not accumulate measurable levels of DIF-I, a morphogen that was previously implicated in prestalk differentiation. In an independent experimental system, wild-type cells respond to the addition of DIF-I by induction of the prestalk marker ecmA and repression of the prespore marker cotB. We found that DIF-1 did not affect the expression of the tagB or carB genes, both of which are prestalk specific and essential for PST-A cell differentiation. We conclude that the initiation of prestalk development is not dependent on DIF-1 and suggest that the morphogen participates mainly at later stages.