In this study, two Pichia methanolica alcohol oxidase (AOD) promoters, PMOD1 and PMOD2, were evaluated in a promoter assay system utilizing the acid phosphatase (AP) gene from Saccharomyces cerevisiae (ScPHO5) as a reporter. Heterologous gene expression driven by the PMOD1 and PMOD2 promoters was found to be strong and tightly regulated by carbon source at the transcriptional level. PMOD1 was induced not only by methanol but also by glycerol. PMOD2 was induced only by methanol, although it was not repressed on the addition of glycerol to a methanol medium, suggesting that PMOD2 is regulated in a manner distinct from that of other AOD-gene promoters. On the other hand, methanol and oxygen level-influenced gene expression mediated by PMOD1 and PMOD2. PMOD1 expression was optimal at low methanol concentrations, whereas PMOD2 was predominantly expressed at high methanol and high oxygen concentrations. Based on these results, both PMOD2 and PMOD1 should be useful tools for controlling heterologous gene expression in P. methanolica. In particular, it should be possible to differentially control the production phases of two heterologous proteins, using PMOD1 and PMOD2 in the same host cell and in the same flask. Copyright © 2006 John Wiley & Sons, Ltd.