Erythropoietin (Epo) gene expression in kidney and liver is inducible by anemia. To localize the sequences necessary for regulated expression of the Epo gene, we constructed transgenic mice containing five human Epo gene constructs and examined Epo expression under basal conditions and with anemia. Mice containing the Epo gene with 0.3 kb of 5' flanking sequence, 0.7 kb of 3' flanking sequence, and either all introns or only intron I alone were polycythemic, had Epo expression in various tissues (including non-Epo-producing tissues), and induction only in liver. In contrast, mice containing the Epo gene with 8.5 kb of 3' flanking sequence and either 9.5 or 22 kb of 5' flanking sequence had basal expression at low levels in appropriate tissues and were less likely to be markedly polycythemic. Mice with the smaller of these two constructs had induction only in the liver, whereas those with the larger construct had induction in the kidney and liver. These studies indicate that sequences sufficient for induction in the liver are located in close proximity to the Epo gene, including the immediate 5' and 3' flanking sequence and the first intron. They also indicate that sequences required for induction in the kidney are located more than 9.5 kb 5' to the gene. Furthermore, comparison of these and prior transgenic studies suggest that sequences that limit the basal expression of the Epo gene are located downstream of the gene. We conclude that multiple cis DNA sequences are required for regulated Epo gene expression.