Pichia pastoris is an excellent host for high-level heterologous gene expression, but there is still much interest in improving the productivity of recombinant protein production. P. pastoris produces a small amount of ethanol as a by-product during the glycerol fed-batch phase and the mixed-feed induction phase (glycerol-methanol) of high cell density fermentations, regardless of the phenotype (Mut(+), Mut(s), or Mut(-)). We have investigated ethanol repression of the AOX1 promoter using strains, GS115 (Mut(+)) and MC100-3 (Mut(-)), expressing an AOX1-lacZ fusion. The addition of 10 mg l(-1) ethanol at the start of methanol induction delayed beta -galactosidase production and methanol utilization for four hours in shake flask experiments. When ethanol and acetate were added together, all of the ethanol was converted to acetate, which also represses the AOX1 promoter. The effects of ethanol and acetate on protein expression in P. pastoris at shake flask and fermentor conditions are discussed.