The effect of platelet lysate on the growth of colonies of human tumor cells was examined in vitro by using the human tumor stem cell assay. Lysates were prepared from freeze-thawed outdated platelets. Eighteen to 446 colonies (mean 85) grew from 211 of 299 tumors plated at a density of 5 X 10(5) cells/plate. The plating efficiency ranged from 0.001% to 0.35%. The viability of the cells plated ranged from 10% to 90%. Platelet lysate significantly enhanced (p less than 0.05) colony formation from cells from 99 (47%) of 211 patients. The number of colonies grown with lysate from these 99 tumors ranged from 36 to 580 (mean 152). The enhancement factor varied from 1.25 to 6.69. Platelet lysate significantly decreased colony formation (p less than 0.05) from cells from 27 (13%) of the 211 tumors and had no effect on 85 (40%). Maximum increases over control occurred after 6 days in 79 (94%) of 84 tumors and after 10 days in 5 (6%). The extent of enhancement by platelet lysate of tumor colony formation correlated with cell viability (r = 0.545, p less than 0.005). The growth-promoting activity for human tumors was nondialyzable, stable with heating at 56 degrees or 70 degrees C for 30 min and at 100 degrees C for 2.5 min, substantially inactivated by heating at 100 degrees C for 5 min, and totally inactivated by heating at 90 degrees C for 30 min. It was partially inactivated by trypsin and abolished by periodate oxidation. The results suggest that a proportion of tumors of epithelial and mesodermal origin are responsive in vitro to growth-promoting factor(s) derived from human platelets.