Increased expression of recombinant mini-proinsulin in Pichiapastoris in 2.51 bioreactors was achieved by increasing the cultivation pH from 5.1 to 6.3, by decreasing the temperature from 28 to 22 degreesC, and by periodical addition of ammonium sulfate and EDTA to the culture broth. Using this procedure, mini-proinsulin reached nearly 0.3 g 1(-1) in the culture supernatant after 160 It of growth.