Cyclophosphamide (CTX) is often used to create a "window" for more effective therapeutic tumor vaccination. According to a commonly applied protocol, we injected 2 mg CTX intraperitoneally to mice with small (2 to 3 mm diameter) or large (5 to 7 mm, and in one experiment 8 to 10 mm diameter) subcutaneously growing tumors from the SW1 clone of the K1735 melanoma, euthanized the mice 4 days later and studied the composition of lymphoid cells by flow cytometry in both spleens and tumors. Administration of CTX increased the percentage of CD3, CD4, and CD8 cells with the increase in tumors being significantly greater than in spleens, and it also increased the percentage of B cells in spleens and tumors. Furthermore, CTX dramatically increased the frequency of tumor-infiltrating CD4 and CD8 cells containing interferon gamma, of cells expressing NK1.1, and of cells expressing the dendritic cell markers CD11c, CD80, and CD86, with the greatest increases seen among tumor-infiltrating lymphoid cells (TIL) from mice with small tumors. Although CTX decreased the percentage of TIL that expressed CD4 or CD8 together with CD25 and FoxP3 and were therefore considered to be regulatory T cells, it increased the frequency of TIL that stained for Gr1/CD11b, a marker for myeloid-derived suppressor cells. We conclude that the administration of CTX can favorably impact several cell populations that are involved in tumor rejection. However, since CTX has a limited effect on TIL from tumors larger than a few millimeter in diameter and in view of an increased percentage of myeloid-derived suppressor cells among TIL from mice given CTX there is a need for more effective ways to improve tumor vaccination.