Using J2E cells and the murine beta maj-globin promoter as a model, we have performed the first direct analyses of erythropoietin (EPO)-activated transcription from defined templates. The -346 to +26 beta maj promoter was shown to comprise a target for maximal activation. This included a positive role for a -346 to -107-base pair (bp) domain in J2E cells, but not in F-MEL cells. Mutagenesis of a -215-bp AGATAA element within this domain showed that this effect did not require GATA-1 binding. In contrast, a critical role for GATA-1 at a -60-bp (G)GATAG element was defined by mutagenesis (GGg-TAG and TGATAG), complementation with a synthetic TGATAA element, and the demonstrated specific binding of GATA-1. Proximal CCAAT (-75) and CACCC (-90) elements also were shown to contribute to transcriptional activation in J2E cells, yet exerted quantitatively distinct effects in the F-MEL system. Based on these results, minimal [TGATAA]4-TATA and TGATAA-CACCC-TATA promoters were constructed and assayed in each system. Remarkably, the [TGATAA]4-TATA promoter, but not the TGATAA-CACCC-TATA promoter, was induced efficiently by EPO in J2E cells, whereas the TGATAA-CACCC-TATA promoter was highly induced by Me2SO in F-MEL cells. These findings suggest that mechanisms of EPO-induced transcription in J2E cells involve GATA-1 and differ from chemically activated mechanisms studied previously in F-MEL cells. Globin induction in J2E cells was not associated with effects of EPO on levels or nuclear translocation of GATA-1. However, hemoglobinization was induced by okadaic acid, 8-Br-cAMP, and forskolin, a finding consistent with induction mechanisms that may involve modulated serine/threonine phosphorylation.