GM-CSF signals through JAK2 and STAT5 and stimulates the expression of STAT5 target genes, such as pim-1 and CIS. Analyzed by EMSA, GM-CSF stimulation led to much stronger STAT5 DNA-binding to pim-1 or CIS GAS elements in primary human monocytes compared with mature macrophages. Similarly, GM-CSF-induced expression of pim-1 and CIS mRNAs was much stronger in monocytes. These differencies were not a result of downregulation of the GM-CSF receptor system or STAT5 expression, because monocytes and macrophages readily expressed GM-CSF receptor, JAK2, STAT5A, and STAT5B mRNAs and proteins. Monocytes expressed significant amounts of truncated STAT5 forms that took part in STAT5-DNA complex formation in GM-CSF-stimulated monocytes. This resulted in faster moving STAT5 complexes compared with macrophages in EMSA. Our results demonstrate that STAT5 isoform expression, GM-CSF-induced STAT5 activation, and STAT5 target-gene expression are altered significantly during monocyte/macrophage differentiation.