Multiple genetic alterations, including inactivation of the RB gene, occur commonly in small-cell lung carcinoma (SCLC). To assess a functional role of RB inactivation in the development of SCLC, an RB expression plasmid was introduced by stable transfection into SCLC cell lines, Lu-135 and N417, in which the RB gene was inactivated. Lu-135 and N417 cells transfected with the wild-type RB gene formed G418-resistant colonies twofold less efficiently than those with a mutated RB gene or with the control vector. Intact exogenous wild-type RB genes were detected only in approximately 20% of G418-resistant clones; three of 14 in Lu-135 and three of 16 in N417, respectively. Transcripts from the transfected RB gene were also detected in two of these three clones from Lu-135 and two of three from N417 but the amount of RB mRNA and protein was less than one fifth of that in normal fibroblast cells WI-38. Furthermore, clones with exogenous wild-type RB expression showed either reduced growth rates in culture or suppressed tumorigenicity in nude mice. These findings suggest that functional correction of the RB gene is sufficient to suppress the growth of SCLC cells, even though several other genetic alterations in the cells remain uncorrected.