Aqualysin I is a heat-stable subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. We report the high-level expression of an aqualysin I protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris. The expression of aqualysin I in P. pastoris was carried out using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Pro-aqualysin I (38kDa) as inactive protein was secreted into the medium of shake-flask cultures at a concentration of 1g/L. It was isolated from the culture supernatant by an ammonium sulfate precipitation and one-step anion exchange chromatography in a nearly pure form and was autoproteolytically activiated by heat treatment. A proteolytic activity test indicated that the purified recombinant aqualysin I was properly folded with a specific activity similar to that of the native enzyme. We also explored the possibility of secreting the GAP expressed aqualysin I in P. pastoris by in-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal. However, the levels of secreted pro-aqualysin I particles were approximately 10 times lower, possibly as a consequence of membrane association or to the influence of the alpha-factor secretion signal sequences on the transcription or secretion of aqualysin I. When considering further optimization of the downstream process and culture conditions for high-level production of recombinant aqualysin I by P. pastoris, this expression system is promising for development as an industrial process.