An analogue of human platelet factor 4 (PF4) lacking affinity for heparin was specifically designed to evaluate the importance of this property in the antitumor effects of recombinant PF4. The purified protein, recombinant PF4-241 (rPF4-241), failed to bind heparin but retained the ability to suppress the growth of tumors in mice. Daily intralesional injections of rPF4-241 significantly inhibited the growth of the B-16 melanoma in syngeneic mice without direct inhibitory effects on B-16 cell growth in vitro. Similar antitumor effects were observed with the human colon carcinoma, HCT-116, grown in nude mice, indicating that the inhibitory activity was neither tumor-type specific nor T-cell dependent. rPF4-241 inhibited endothelial cell proliferation in vitro with dose dependence similar to the native sequence rPF4. Both rPF4 and rPF4-241 inhibited angiogenesis in the chicken chorioallantoic membrane. The analogue, however, was inhibitory at lower concentrations than rPF4 in the chorioallantoic membrane system and its inhibitory effects were not abrogated by the presence of heparin. The present findings support the conclusion that both rPF4 and rPF4-241 inhibit tumor growth by suppression of tumor-induced neovascularization. The finding that this activity is independent of heparin binding may allow the development of PF4-based angiostatic agents with reduced toxicity and improved bioavailability. These results also suggest that PF4 may play a more specific role in modulation of blood vessel development than previously recognized.