Caffeine is a well known base analogue and is cytotoxic to both animal and yeast cells. There are two possible mechanisms by which yeast cells tolerate caffeine concentrations higher than normal, by mutation or by physiological adaptation. We have isolated novel caffeine-resistant mutants of S. pombe which define three distinct genes caf2, caf3 and caf4. These mutants achieved a level of caffeine resistance which is presumed to represent the upper limit attainable by mutation. The caf2-caf4 mutations, as well as the previously identified caf1 mutation, confer UV-sensitivity, caffeine-resistant UV repair, impaired fertility and sporulation, as well as a lengthened cell cycle. They are partially dominant for caffeine resistance and recessive for UV sensitivity. Some auxotrophic caf3-89 double mutants show drastically decreased caffeine resistance. The caf4 mutant is more resistant to gamma-radiation than wild-type cells and shows pH-sensitive growth. As each caf mutation can, individually, confer maximum caffeine resistance to the cells, all four genes are expected to operate in the same pathway. This pathway might also be responsible for the physiological adaptation since adaptation is lost in caf1-caf4 mutants.