In this study, we examined the effect of oxygen delignification on the rate and extent of enzymatic hydrolysis (using commercial cellulase and P-glucosidase) of a number of lignocellulosic substrates, including kraft pulp (model substrate), pulp mill primary clarifier sludge (PCS) and steam-exploded Douglas fir chips. Oxygen delignification removed up to 67% of the lignin from softwood pulp and improved the rate of, and yield from, hydrolysis by up to 111% and 174%, respectively. Glucose yield varied linearly with fractional lignin removal. Oxygen delignification of primary clarifier sludge improved hydrolysis yield by up to 90%. However steam-exploded Douglas fir was very resistant to hydrolysis at low enzyme loading, and oxygen delignification decreased hydrolysis rate and yield.