The effects of butyrate, retinol, and retinoic acid were tested on growth and differentiation of human Y-79 retinoblastoma cells in monolayer cultures. Treatment with 4 mM butyrate resulted in marked growth inhibition of cells, due mostly to increased death rate. The effect was greater in serum-supported cultures in which the proliferation rate was high and the effect was less in the serum-free, defined medium in which the cells were differentiated and the growth rate was slow, suggesting a cell-cycle-specific action of this substance. Moreover, butyrate induced morphologic changes in the viable cells; these changes consisted of an elongated appearance of the cells and of retraction of long processes formed in the serum-free-supported cultures. Retinol at 20 microM also affected the cell viability both in serum-containing and in serum-free culture medium. Retinoic acid at 50 microM induced reversible growth inhibition of cells growing in serum-containing medium and cell death in the defined medium-supported cultures. Combination of 0.5 mM butyrate with 50 microM retinoic acid resulted in an enhanced inhibition of growth in an apparently synergistic fashion. These results demonstrated that butyrate, retinol, and retinoic acid suppressed Y-79 cell growth in vitro and could be useful in future studies of these compounds in retinoblastoma tumors in vivo.