Human carcinoma HEp-3 lost its tumorigenic and metastatic potential upon prolonged culture in vitro. This change was accompanied by a reduced production of plasminogen activator (PA) of the urokinase type (uPA), which is secreted by HEp-3 cells, a change in response to effectors that modulate uPA production, and an alteration of cell morphology. Similar but more rapid changes occurred when malignant HEp-3 cells were exposed to dimethyl sulfoxide (DMSO). uPA activity in the culture medium dropped below 50% of the control level within 6 h after the addition of DMSO and became undetectable after 24 h of treatment. This drop in uPA activity was not caused by an increased production of PA inhibitors. The cell-associated uPA decreased to 25 to 30% of the control level within 6 h of DMSO treatment and remained at this level for at least 96 h; the reduced uPA production was partially accounted for by a rapid decrease in the functional and chemical concentration of uPA mRNA. In contrast, the concentrations of most of the abundant mRNA species did not appear to be significantly affected, and cell growth was only slightly inhibited in the presence of DMSO. Malignant HEp-3 cells treated with DMSO responded to cholera toxin with an enhanced production of uPA, and their morphology became indistinguishable from that of nonmalignant HEp-3 cells grown in vitro for prolonged periods of time. All of the above changes were fully and rapidly reversible. The inhibitory effect of DMSO on PA production appears to be specific for uPA of human cell lines.