The antiproliferative and cytodifferentiating effects of a new stable butyric derivative, monobut-3, were compared using human MDA-MB-231 breast cancer cells grown in three dimension as either in vitro tumor nodules or in vivo xenograft tumors. In in vitro tumor nodules, monobut-3 exhibited marked growth inhibitory effects consistent with the results obtained in monolayer cell cultures. Some functional cell differentiation was also detected in treated nodules. In in vivo xenografts, monobut-3 significantly decreased MDA-MB-231 tumor take but did not affect the rate of tumor growth. No difference was noted in the histological characteristics of the xenografts between untreated and treated mice. Moreover, once monobut-3 treatment was discontinued, tumor growth rapidly resumed in tumor-free animals. The decreased efficacy of monobut-3 in in vivo MDA-MB-231 xenografts as compared to in vitro tumor nodules indicates that factors related to host environment may still limit the clinical effectiveness of this compound.