The methylotrophic yeast Pichia pastoris synthesizes high levels of alcohol oxidase from the AOX1 gene during growth on methanol as a carbon source. We have a transcriptional fusion of the lacZ gene to the AOX1 promoter as a model system for investigating recombinant protein production in an alcohol oxidase (aox1, aox2) defective strain. Growth and recombinant protein production with glycerol as the carbon source (fed at various constant feedrates) was studied. A feedrate of 1 g l(-1) h(-1) was found to be optimum resulting in a specific activity of 8.62 x 10(4) U mg(-1) dry cell. The specific yield did not improve when glycerol was increased in steps. High feedings rates gave low specific yields (U mg(-1) dry cell mass) and high cell masses. Low protein yields at higher glycerol feedrates were due to partial repression of the AOX1 promoter by glycerol and the by-product, ethanol. In comparison, the wild type (Mut(+)) strain gave a maximum specific yield of 5.52 x 10(4) U mg(-1) dry cell.