The anti-proliferative effects of pivaloyloxymethyl butyrate (AN-9), a butyric acid (BA) derivative with potent tumor-differentiating properties both in vitro and in vivo, was evaluated against colorectal, breast, lung, ovarian, renal cell, bladder, and other types of tumor colony-forming units in a human tumor cloning assay. A total of 76 evaluable specimens were exposed to AN-9 continuously, 48 of these were also exposed to BA continuously for direct comparison of the two agents, and 20 specimens were exposed to AN-9 for two hours. An in vitro inhibitory response was defined as a > or = 50% decrease in tumor colony formation in treated cells compared to untreated controls. Superior anti-tumor activity was observed with the continuous exposure to AN-9 (39% in vitro response at 100 microM and 70% at 200 microM) than with the two-hour exposure (20% at 100 microM and 25% at 200 microM). At a continuous concentration of 200 microM, AN-9 demonstrated greater tumor-specific activity than BA against melanoma (100% vs. 67%), ovarian (67% vs. 40%), breast (63% vs. 0%), non-small cell lung (60% vs. 10%), and colorectal tumor colony-forming units (62% vs. 20%). AN-9 is a novel differentiating agent with activity against colony-forming units derived from a variety of primary human tumors, including those that are considered relatively chemoresistant, and may thus provide a therapeutic alternative or addition to standard cytotoxic agents, if appropriate drug concentrations can be achieved in patients.