WERI-Rb27 human retinoblastoma cells were reconstituted with an intact RB gene by retrovirus-mediated gene transfer, in order to study the phenotypic effects of the protein in vitro and in vivo. Extensive morphological changes were observed, dominated by the formation of multinucleated giant cells. Six weeks after retroviral infection, the giant cells began to die and small cells emerged, resembling the parental non-reconstituted line. They expressed RB and continued to grow, although they showed an increased sensitivity to serum starvation. The original RB-negative cells grew progressively after subcutaneous inoculation into SCID mice, whereas the reconstituted cells failed to grow. RB-positive cells grew progressively in the corpus vitreum of the eye and in the brain, however. The RB-reconstituted cells grew more slowly and were less invasive than the parental cells and cells infected with a firefly luciferase (LUX) gene carrying retrovirus, used as controls. RB-reconstituted cells re-explanted from the intraocular and intracranial tumors continued to express full-length RB protein. RBeye2, an RB-positive cell line established from an eye tumor, was still unable to grow subcutaneously. The reduced tumorigenicity of the RB-reconstituted cells in the subcutaneous space may be due to the influence of locally acting growth-controlling signals or the absence of microenvironment-specific trophic factors. Alternatively, it may reflect the action of residual immune effectors in the SCID mice. If this is the case, these would have to be more effective at the subcutaneous site than in the eye or brain.