We studied peripheral blood lymphocytes (PBL) from 42 patients with metastatic melanoma undergoing treatment with cyclophosphamide (CY) plus melanoma vaccine to determine whether CY immunopotentiation could be related to depletion of T-cells that function as inducers of suppression. Every 28 days, the patients were given CY, 300 mg/m2 i.v., followed 3 days later by the intradermal injection of autologous, irradiated melanoma cells mixed with Bacillus Calmette-Guérin. PBL were separated by density gradient centrifugation and cryopreserved until needed for testing. They were stained with monoclonal antibodies directly conjugated to fluorescein isothiocyanate or phycoerythrin and analyzed by two-color flow cytometry. At no time after the initiation of CY plus vaccine were there any significant changes in the percentages of helper-inducer T-cells (CD4+), suppressor-cytotoxic T-cells (CD8+), or the subpopulation of CD8+ cells expressing Leu 15, a marker for suppressor cells. Treatment of melanoma patients with CY plus vaccine resulted in a progressive fall in the proportion of CD4+ T-cells expressing the 2H4 (CD45) antigen, which identifies inducers of suppression. The reduction of CD4+, 2H4+ T-cells did not become apparent until day 28 after the first dose of CY and reached statistical significance only on days 49 (21 days after the second dose) and 105 (21 days after the fourth dose) (mean changes +/- SE: day 49, -5.4 +/- 1.4%, P less than 0.01; day 105, -9.1 +/- 2.2%, P less than 0.01; t test for nonindependent samples). In contrast, the proportion of CD4+ T-cells expressing the antigen 4B4 (CDw29), which are true helper cells, increased slightly, although not significantly, following the institution of CY plus vaccine (mean changes: day 49, +2.9 +/- 2.1%; day 105, +3.6 +/- 2.4%). Similar results were obtained when absolute numbers of circulating cells, rather than percentages, were analyzed. Thus the number of CD4+, 2H4+ T-cells fell from a mean of 395,000/ml on day 0 to 309,000/ml on day 49 (P less than 0.01) to 256,000/ml on day 105 (P less than 0.05). The absolute number of CD4+, 4B4+ cells remained unchanged at the same time points. These changes were not due to progression of metastatic disease, since a comparison of patients with progressive metastases with those who were rendered disease free by surgery showed no significant differences in the reduction of the percentage of CD4+, 2H4+ T-cells.(ABSTRACT TRUNCATED AT 400 WORDS)