Objectives To characterize the genes responsible for ethanol utilization in Pichia pastoris. Results ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Delta adh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Delta adh3 Delta adh) also produced ethanol. The Adh activities of X33 and MK116 (Delta adh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism. Conclusion The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.