Chemotherapy is assumed to be immunosuppressive; yet to the authors' knowledge, the effects of common chemotherapy protocols on adaptive immune responses in dogs with cancer have not been fully evaluated. Therefore, a study was conducted to evaluate the effects of 2 common chemotherapy protocols on T- and B-cell numbers and humoral immune responses to de novo vaccination in dogs with cancer. Twenty-one dogs with cancer (12 with lymphoma, 9 with osteosarcoma) were enrolled in a prospective study to assess effects of doxorubicin versus multi-drug chemotherapy on adaptive immunity. Numbers of circulating T and B cells were assessed by flow cytometry, and antibody responses to de novo vaccination were assessed before, during, and after chemotherapy. The T- and B-cell numbers before treatment also were compared with those of healthy, age-matched, control dogs. Prior to treatment, dogs with cancer had significantly fewer (P < .05) CD4+ T cells and CD8+ T cells than did healthy dogs. Doxorubicin treatment did not cause a significant decrease in T- or B-cell numbers, whereas treatment with combination chemotherapy caused a significant and persistent decrease in B-cell numbers. Antibody titers after vaccination were not significantly different between control and chemotherapy-treated dogs. These findings suggest that chemotherapy may have less impact on T-cell numbers and ability to mount antibody responses in dogs with cancer than was previously anticipated, though dogs with lymphoma or osteosarcoma appear to be relatively T-cell deficient before initiation of chemotherapy.