Background: Regulation of gene expression by signal transducer and activator of transcription 1 (STAT1) within host tissues mediates the antitumor effects of interferon alfa (IFN α). We used a novel flow cytometric assay to examine phosphorylation-mediated activation of STAT1 within immune effector cell subsets following in vitro or in vivo IFN α treatments. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 17) or melanoma patients (n = 19) were treated in vitro with interferon alfa-2b (IFN α-2b) or phosphate-buffered saline (PBS) and subjected to multiparametric flow cytometry to measure the levels of phosphorylated STAT1 (P-STAT1) within immune cell subsets. We similarly analyzed PBMCs isolated from melanoma patients before and 1 hour after immunotherapy with IFN α-2b. All statistical tests were two-sided. Results: P-STAT1 levels in all major immune cell subsets increased within 15 minutes of in vitro IFN α-2b treatment of PBMCs; the increase was most pronounced in T lymphocytes and monocytes. Relatively low doses of IFN α-2b (i.e., 10 ² –10 ³ IU/mL) induced maximal STAT1 activation in vitro . Compared with melanoma patients, healthy donors had higher basal levels of P-STAT1 (specific fluorescence [Fsp]; i.e., Fsp _PBS , the level of P-STAT1 in PBS-treated cells) in total PBMCs, natural killer (NK) cells, and T cells (mean Fsp _PBS in total PBMCs: 5.5 in healthy donors versus 1.6 in patients, difference = 3.9, 95% confidence interval [CI] = 1.4 to 6.5, P = .004; mean Fsp _PBS in NK cells: 4.6 in healthy donors versus 0.9 in patients, difference = 3.7, 95% CI = 1.7 to 5.7, P = .001; mean Fsp _PBS in T cells: 6.8 in healthy donors versus 0.9 in patients, difference = 5.9, 95% CI = 2.5 to 9.3, P = .002). P-STAT1 was detected in the NK and T cells of two patients who received IFN α-2b immunotherapy (20 MU/m ² [MU = million units], administered by intravenous injection). P-STAT1 levels in the PBMCs of a patient treated sequentially with 5 MU/m ² and 10 MU/m ² IFN α-2b (administered by subcutaneous injection) also increased in response to treatments with IFN α-2b but did not increase further with the increased dosage of IFN α-2b. Conclusion: This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN α immunotherapy.