Neutropenic patients suffering from gynecologic cancers treated with cancer chemotherapy were evaluated to investigate changes in levels of plasma alkaline phosphatase (ALP) during recombinant human granulocyte colony stimulating factor (rhG-CSF) administration, and to discern its causes. Plasma ALP, ALP isozymes, neutrophil alkaline phosphatase (NAP) activity, and morphological maturation of neutrophils were measured prior to, during, and after rhG-CSF administration. Plasma ALP values were significantly elevated (p < 0.05) during administration of rhG-CSF. ALP-3, the dominant alkaline phosphatase fraction of NAP, was the dominant isozyme that showed an increase in the plasma. Moreover, the elevation of plasma ALP-3 significantly correlated with the elevation of NAP activity (r = 0.939, p < 0.0001), and neutrophils in which NAP activity was induced were not limited to morphologically mature neutrophils. These results showed that rhG-CSF acts to stimulate neutrophils at all stages of maturation and causes an elevation of plasma ALP(ALP-3) concentrations in plasma.