Heterozygous inactivation of either human TSC1 or TSC2 causes tuberous sclerosis (TSC), in which development of benign tumors, hamartomas, occurs via a two-hit mechanism. In this study, fission yeast genes homologous to TSC1 and TSC2 were identified, and their protein products were shown to physically interact like the human gene products. Strains lacking tsc1(+) or tsc2(+) were defective in uptake of nutrients from the environment. An amino acid permease, which is normally positioned on the plasma membrane, aggregated in the cytoplasm or was confined in vacuole-like structures in Deltatsc1 and Deltatsc2 strains. Deletion of tsc1(+) or tsc2(+) also caused a defect in conjugation. When a limited number of the cells were mixed, they conjugated poorly. The conjugation efficiency was improved by increased cell density. Deltatsc1 cells were not responsive to a mating pheromone, P-factor, suggesting that Tsc1 has an important role in the signal cascade for conjugation. These results indicate that the fission yeast Tsc1-Tsc2 complex plays a role in the regulation of protein trafficking and suggest a similar function for the human proteins. We also show that fission yeast Int6 is involved in a similar process, but functions in an independent genetic pathway.