The expression of the major erythroid DNA-binding protein GATA-1 was studied during erythropoietin and chemically induced erythroid differentiation of J2E and murine erythroleukemia (MEL) cells. An increase in GATA-1 mRNA levels was observed shortly after hormonal stimulation of J2E cells, due to increased transcription and not stabilization of the short-lived transcripts. Concomitantly, an increase in protein capable of binding to the GATA motif was detected. Premature removal of erythropoietin from culture resulted in submaximal GATA-1, globin, and hemoglobin production. In contrast, differentiation of J2E cells initiated by sodium butyrate resulted in a sudden depletion of GATA-1 transcripts. Similarly, dimethyl sulphoxide induction of MEL cells produced a transient decrease in GATA-1 mRNA. Surprisingly, these decreases in mRNA were not reflected in alterations to GATA-1 protein content, or the ability of the transcription factor to bind DNA. We concluded from this study that the sequence of events initiated by erythropoietin was not followed during chemical stimulation of erythroid differentiation.