The cytochrome P450alk gene (P45alk) from Candida tropicalis ATCC 750 was expressed in Saccharomyces cerevisiae GRF18 under control of the alcohol dehydrogenase I (ADHI) promoter. To achieve stable expression over long time periods, a 2-microns derived replicative and an integrative expression system were tested in continuous culture. The 2-microns derived replicative system could not be maintained in cells over high generation numbers. In continuous culture, the instability was more pronounced at high dilution rates (D) and high histidine concentration, for which the yeast is auxotrophic. The nature of the instability was probably due to a gene conversion event between the plasmid and the yeast chromosome. In contrast, the integrative expression system was stably maintained in cells over prolonged cultivation times. Since this work focused on the production of large quantities of P450 by heterologous expression in yeast over prolonged time periods, the integrant was used to optimize P450alk expression by varying continuous culture parameters. The P450alk expression was shown to be dependent on the D applied to the culture. The highest P450alk expression levels were obtained at high D, when cell metabolism was shifted to partial glucose oxidation, yielding ethanol as a major metabolite in the culture supernatant. In contrast, when glucose was completely oxidized at low D, the ADHI-dependent P450alk expression was reduced and followed by a corresponding decrease in heterologous protein.