Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates many of the biological functions of human neutrophils. This includes the stimulation of protein synthesis and the tyrosine phosphorylation of various proteins among which is JAK2. The present study was aimed at characterizing in detail the pattern of activation by GM-CSF of the JAK/STAT pathway in human neutrophils. The results obtained show that the stimulation of human neutrophils by GM-CSF specifically led to tyrosine phosphorylation of JAK2 and had no effect on JAK1, JAK3, or TYK2. Furthermore, GM-CSF induced the tyrosine phosphorylation of STAT3 and STAT5 but not of STAT1, STAT2, STAT4, or STAT6. Tyrosine phosphorylation of STAT3 was transient reaching its maximum at 15 min. STAT5 presented a different pattern of tyrosine phosphorylation. The anti-STAT5 antibodies identified two proteins at 94 and 92 kDa. The 94-kDa STAT5 was constitutively tyrosine phosphorylated and showed no change upon GM-CSF stimulation. On the other hand, the 92-kDa STAT5 was tyrosine phosphorylated within 1 min of GM-CSF treatment and this was maintained for at least 30 min. By the use of specific antibodies, it was determined that only STAT5B, and not STAT5A, was tyrosine phosphorylated in GM-CSF-treated neutrophils. Furthermore, GM-CSF treatment induced an increase in the ability of STAT3 and STAT5B, but not STAT5A, to bind DNA probes. The specificity of the pattern of activation of the JAK/STAT pathway suggests that it may be directly linked to the modulation of the functions of mature nondividing, human neutrophils by GM-CSF.