Endostatin is a 20 kDa carboxyl-terminal fragment of collagen XVIII that strongly inhibits angiogenesis and tumor growth. The methylotrophic yeast, Pichia pastoris, is a robust expression system that can be used to study methods to improve the yields of rhEndostatin. We expressed rhEndostatin in P. pastoris under the control of the alcohol oxidase 1 (aox 1) promoter (Mut(+) phenotype) as a model, and used a cell biomass of about 50 g l(-1) dry cell wt as a starting point for the induction phase and varied the methanol feed rate at 8 ml l(-1) h(-1), 11 ml l(-1) h(-1) and 15 ml l(-1) h(-1). While the cell growth rate was proportional to the rate of methanol delivery, protein production rate was not. These findings could be used to guide parameters for large-scale production of recombinant proteins in the P. pastoris system.