Binding of erythropoietin (Epo) to the Epo receptor (EpoR) initiates a signaling cascade resulting in tyrosine phosphorylation of several proteins and induction of AP-1 transcription factor(s). While Epo is known to activate c-fos gene expression, the mechanism of AP-1 activation is unknown. Here we show that AP-1 activation by Epo requires tyrosine kinase activity and also de novo protein synthesis. Using a mutant EpoR containing no cytosolic tyrosine residues, and a set of eight mutants containing a single cytosolic tyrosine residue, we show that multiple EpoR tyrosines, thought to activate multiple intracellular signal transduction proteins, can mediate AP-1 activation. An EpoR containing only tyrosine 343 or tyrosine 464 supports a maximal level of AP-1 activation. We also show that AP-1 activation does not require maximal STAT5 activation and may occur via a STAT5-independent signaling pathway. JOURNAL OF BIOLOGICAL CHEMISTRY .