Rsu-1, which was isolated based on its ability to suppress transformation by v-Ras, is a highly conserved gene which shares homology with yeast adenylyl cyclase in the region required for activation by Ras. Genomic DNA clones of human RSU-1 have been isolated and used as a probe for fluorescence in situ hybridization (FISH) to assign RSU-1 to 10p13, confirming the previous results of somatic cell hybrid mapping localizing RSU-1 to chromosome 10. Screening of more than 20 human tumor cell lines for RSU-1 expression revealed that most cell lines contained abundant RSU-1 RNA and protein. However, the p33 RSU-1 protein was undetectable in the U251 glioblastoma cell line and transfection of a rsu-1 expression vector into U251 cells yielded a cell line in which rsu-1 was under the control of a regulatable metallothionein promoter. Addition of Cd2+ to the U251-Rsu-1 transfectant resulted in transcription of rsu-1 RNA and the accumulation of p33 Rsu-1 protein. Appearance of the Rsu-1 protein correlated with a reduction in growth rate of the U251-Rsu-1 transfectant. In addition, reduction in anchorage independent growth and phenotypic alteration in U251-Rsu-1 transfectant agar colonies was observed. Two U251-Rsu-1 transfectant cell lines were non tumorigenic when injected subcutaneously into athymic nude mice. These results, in conjunction with the frequent deletions observed in chromosome 10 in glioblastomas, suggest that RSU-1 loss of function may play a role in the progression of this disease.