There is a large body of evidence suggesting the connexin gap junction proteins appear to act as tumor suppressors, and their tumor inhibitory effect is usually attributed to their main function of cell coupling through gap junctions. However, some cancer cells (e.g. the rat bladder carcinoma BC31 cell line) are cell-cell communication proficient. Using specific site-directed mutagenesis in the third membrane-spanning (3M) domain of connexin43 (Cx43), we abolished the intrinsic gap junction intercellular communication (GJIC) in BC31 cells either by closing the gap junctional channels or by disruption of the transport of connexin complexes to the lateral membrane. Clones of BC31 cells transfected with a dominant negative Cx43 mutant giving rise to gap junctional channels, permeable only for a small tracer (neurobiotin), displayed accelerated growth rate in vivo, showing the critical role of selective gap junctional permeability in the regulation of cell growth in vivo. The use of other dominant-negative mutants of Cx43 also suggested that the effect of impaired communication on the tumorigenicity of cancer cells depends on the subcellular location of connexin. Inhibition of intrinsic GJIC in BC31 cells by sequestering of Cx protein inside the cytoplasm, due to expression of dominant-negative transport-deficient Cx43 mutants, did not significantly enhance the growth of transfectants in nude mice, but occasionally slightly retarded it. In contrast, augmentation of GJIC in BC31 cells by forced expression of wild-type Cx43, or a communication-silent mutant, fully suppressed tumorigenicity of these cells. Overall, these results show that cell coupling is a strong, but not the sole, mechanism by which Cx suppresses growth of tumorigenic cells in vivo; a GJIC-independent activity of Cx proteins should be considered as another strong tumor-suppressive factor.