STAT5a and STAT5b are two highly related transcription factors that control essential cellular functions. Several STAT5 targets are known, although it is likely that most remain uncharacterized. To identify a more complete set of STAT5-regulated genes, we used a modification of the chromatin immunoprecipitation procedure, which does not presuppose any information regarding these targets. Employing Ba/f3 cells in which STAT5 is activated by interleukin-3, we have identified novel STAT5 binding sites that may be regulatory regions for nearby genes. These sites are typically found far from transcription start sites, and most do not contain CpG islands, indicating that they are not in traditional promoter regions. Nonetheless, when the expression of genes near these STAT5 binding sites was examined, all were expressed in Ba/f3 cells, and most were modulated by interleukin-3. Furthermore, genes identified by this strategy show unique expression patterns in acute leukemias, tumors characterized by activated STAT5. Whereas both STAT5 isoforms bound to all promoters tested, STAT5a and STAT5b bound with different kinetics, suggesting that at least some of the differences between the functions of these two proteins are mediated by their DNA binding activity. Therefore, this method of transcription factor target identification represents an effective strategy to isolate transcription factor targets in an unbiased fashion, and it has revealed many novel STAT5-dependent regulatory regions outside of traditional promoters.