The morphological, cultural and biochemical characteristics of 80 M. pachydermatis strains isolated from cases of canine otitis externa were studied. Microscopically, the strains could be subdivided into two phenotypes. All M. pachydermatis strains grew well on Sabouraud glucose, yeast morphology and modified malt extract agar, but formed two distinct colony types. All strains were characterized by no fermentation. Assimilation of glucose, mannitol (42 strains), sorbitol (40 strains) and peptone was observed, but no ethanol assimilation. Urease and catalase tests were positive, while indole and acetoin production was not detected. All strains showed proteinase, caseinase, lecithinase and peroxidase positivity but to varying extents. Esterase activity was observed for all Malassezia strains when using Tween 20, 40 and 60, whereas Tween 80 was hydrolysed by only 42 strains. No coagulase or haemagglutinating activities were detected. When compared for satellite phenomenon and vitamin requirements, some Malassezia strains could not grow in the absence of nicotinic acid but grew well in the presence of staphylococci. In susceptibility tests, all strains showed the highest susceptibility to ketoconazole. On the basis of the biochemical differences, M. pachydermatis seems to be a heterogeneous species and can be divided into two groups.