We have analyzed the function of several mammalian promoter elements in the fission yeast, Schizosaccharomyces pombe. Mutants of the human HSP70 promoter were introduced into S. pombe as single copy integrants at a specific location. Transcription initiation sites utilized in S. pombe with the HSP70 TATA element were similar to those used in mammalian cells. Of three mammalian TATA elements tested, only the HSP70 TATA element functioned in S. pombe. The adenovirus Ella TATA element had little or no activity in S. pombe, indicating that S. pombe is deficient in the factor(s) necessary for recognition of this element. Of upstream promoter elements tested, the CCAAT, Sp1 binding, ATF binding and heat shock elements were functional in S. pombe. Strains containing mutant promoters fused to the ble gene were used to demonstrate that phleomycin can be used as a graded selection in S. pombe. These data demonstrate that S. pombe should provide a useful system in which to characterize and isolate mammalian factors involved in initiation site determination and transcriptional regulation.