We describe the expression and in vitro activity of recombinant tumstatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells. Recombinant tumstatin was secreted into a culture medium with a molecular weight of 29 kDa. Recombinant tumstatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant tumstatin expressed in stably transformed Tn 5B1-4 cells was approximately 0.76 microg/ml. A maximum production level of 4.0 mg/l recombinant tumstatin was obtained in a T-flask culture of Tn 5B1-4 cells, 6 days after cultivation. We also investigated the individual effects of both dimethyl sulfoxide (DMSO) and sodium butyrate on recombinant tumstatin production in stably transformed Tn 5B1-4 cells. Supplementing cultures with DMSO and sodium butyrate separately increased recombinant tumstatin production in stably transformed Tn 5B1-4 cells by 117 and 32%, respectively.