Analysis of fatty acid composition in wild-type cells of Hansenula polymorpha strain CBS 1976 revealed the presence of 18 : 1 (Delta 9), 18:2 (Delta 9,12) and 18:3 (Delta 9,12,15) unsaturated fatty acids (UFAs), indicating that the alpha-linolenic desaturation pathway operates in this yeast. H. polymorpha cells also showed ability for uptake and incorporation of exogenous UFAs. By ethyl methanesulfonate mutagenesis, nine unsaturated fatty acid auxotrophic mutants of H. polymorpha were isolated. These mutants exhibited the growth arrest phenotype on nutrient medium and on nutrient medium supplemented with saturated fatty acids, but grew on media supplemented with various UFAs. Genetic analysis revealed that single recessive nuclear mutation conferred Ufa auxotrophy on these mutants. Fatty acid analysis by gas chromatography showed the accumulation of 18 : 0 but a decrease in the amount of 18:1 and 18:2 in mutant cells compared with the wild-type cells. Integrated physiological and genetical data suggested that mutations in all mutants occurred in one gene and probably led to defects in Delta 9-desaturation pathway.