Saccharomyces carlsbergensis is an amphiploid, and it has previously been suggested that the genomes of S. carlsbergensis originate from S. cerevisiae and S. monacensis. We have cloned the ACB1 genes encoding the acyl-CoA binding protein (ACBP) from S. carlsbergensis, S. cerevisiae and S. monacensis. Two genes were found in S. carlsbergensis and named ACB1 type 1 and type 2, respectively. The type 1 gene is identical to the S. cerevisiae ACB1 gene except for three substitutions, one single base pair deletion and one double base pair insertion, all located in the promoter region. The type 2 gene is completely identical to the S. monacensis ACB1 gene. These findings substantiate the notion that S. carlsbergensis is a hybrid between S. cerevisiae and S. monacensis. Both ACB1 type 1 and type 2 are actively transcribed in S. carlsbergensis and transcription is initiated at sites identical to those used for transcriptional initiation of the ACB1 genes in S. cerevisiae and S. monacensis, respectively. Two polyadenylation sites, spaced 225 bp apart, are present in the S. cerevisiae ACB1 gene. The upstream polyadenylation site is used exclusively during exponential growth, whereas both sites are utilized during later stages of growth.