Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects. These defects are associated with a poor prognosis and are mediated, in part, by immune inhibitory CD34(+) progenitor cells, whose numbers are increased in the peripheral blood of HNSCC patients. Immune inhibitory CD34(+) cells are also present within HNSCC tumors. A phase IB clinical trial was conducted with HNSCC patients to determine if treatment with the differentiation-inducer 25-hydroxyvitamin D(3) could diminish CD34(+) cell levels and improve a panel of immune parameters. Here we present the results of treatment with orally administered escalating doses (20, 40, 60 microg) of 25-hydroxyvitamin D(3), with an emphasis on the six patients who received the maximum dosage of 60 microg per day. Peripheral blood was collected at 0, 1, 2, 4, and 6 weeks, and assessed for markers of immune activity. Although no clinical responses were observed, results of this pilot study demonstrated that treatment of HNSCC patients with 25-hydroxyvitamin D(3 )reduces the number of immune suppressive CD34(+) cells, increases HLA-DR expression, increases plasma IL-12 and IFN-gamma levels, and improves T-cell blastogenesis. In contrast, 25-hydroxyvitamin D(3) treatment did not modulate plasma IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, or TGF-beta levels.