Commercially available oil cakes such as coconut oil cake (COC), sesame oil cake (SOC), palm kernel cake (PKC), groundnut oil cake (GOC), cottonseed oil cake (CSC) and olive oil cake (OOC) were used as substrates for phytase production in solid-state fermentation using three strains of Rhizopus spp., namely Rhizopus oligosporus NRRL 5905, Rhizopus oryzae NRRL 1891 and R. oryzae NRRL 3562. COC was the most preferred substrate, in general, for all the three strains; GOC and PKC resulted in comparable enzyme titers with R. oryzae NRRL 1891 but CSC and OOC poorly supported the cultures in producing phytase. R. oryzae NRRL 1891 produced the highest titers of phytase on COC (30.1 U enzyme per gram dry substrate, U/gds), followed by SOC (28.9 U/gds). Mixed substrate fermentation using COC and SOC in the ratio 1: 1 (w/w) further enhanced enzyme production by R. oryzae NRRL 1891 to 35 U/gds. An incubation time of 72 h, initial moisture of 52% and an inoculum of 1 ml was the optimum cultural conditions for the production of phytase in mixed substrate fermentation. Supplementation of the fermentation medium with 1% glucose increased phytase activity to 52 U/gds. Addition of ammonium nitrate at 0.5% concentration resulted in further enhancement of the enzyme tires to 64 U/gds. Thus, mixed substrate fermentation using COC and SOC resulted in more than two-fold increase in phytase production under optimized conditions (64 U/gds phytase in comparison to 30.1 U/gds by COC individually). Results obtained appear to be of commercial significance showing the potential of oilcakes and mixed substrate fermentation for phytase production. (c) 2004 Elsevier Ltd. All rights reserved.