Four isoforms (alpha, beta, gamma, and delta) have been identified for signal transducer and activator of transcription 3 (STAT3). It has been reported that STAT3gamma, which is derived from STAT3alpha by limited proteolysis during granulocytic differentiation, is a major STAT3 isoform expressed in human neutrophils. We confirmed that STAT3gamma was a major STAT3 isoform detected in human neutrophil lysates prepared with the conventional lysis buffer. The enzymes capable of converting STAT3alpha to STAT3gamma in vitro were localized in neutrophil granule fraction and were released into the medium upon ionomycin stimulation. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, CuSO(4), and ONO-5046 (a specific inhibitor of neutrophil elastase), but not by aprotinin, leupeptin, benzamidine, and EDTA. STAT3gamma was effectively generated in vitro from STAT3alpha by limited proteolysis with human neutrophil elastase or proteinase 3 but not cathepsin G. The converting activity in neutrophil lysates was reduced by immunodepletion of elastase but not proteinase 3. Unexpectedly, STAT3gamma was undetected in the lysates of neutrophil-derived cytoplasts, which lack granules, and the cytosol fraction prepared by nitrogen cavitation. The STAT3 isoform detected in these preparations was primarily STAT3alpha. STAT3gamma was also undetected in the lysates of PMSF-pretreated neutrophils and was markedly decreased in the lysates of ionomycin-pretreated neutrophils. These findings indicate that, in contrast to the previous reports, STAT3alpha, but not STAT3gamma, is primarily expressed in human neutrophils, and STAT3gamma is rapidly generated from STAT3alpha by limited proteolysis with granule-derived serine proteases during preparation of neutrophil lysates with the conventional lysis buffer.