Vaccinia melanoma oncolysates (VMO) were tested in a Southeastern Cancer Study Group (SECSG)-sponsored phase I/II multiinstitutional trial. Forty-eight patients with stage I or II disease were placed on study at six different dose levels of VMO and two different dose schedules, immediate or delayed. Patients' sera, obtained before treatment and every 3 months following initiation of treatment, were tested for antimelanoma antibodies using a Staphylococcus protein A (SpA) assay. Pretreatment sera were negative in 46 of 47 patients, and only two of 19 patients on delayed treatment developed reactivity by 6 months. However, 13 of 23 on immediate treatment developed reactivity, including eight of eight at the higher doses (1.5 and 2.0 mg). Neither anti-HLA antibody tested by a standard microcytotoxicity assay nor circulating immune complexes measured by both Clq and conglutinin binding assays were produced as a result of the immunization. The demonstration of immunogenicity of VMO at the 2 mg dose and immediate schedule supported the rationale for the use of this dose and schedule for the ongoing second phase Ia/Ib trial and for the future phase III randomized prospective study.