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The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain Penicillium sp. in solid-state fermentation (SSF).Certain fermentation parameters involving incubation temperature, moisture content, initial pH value, inoculum and load size of medium, and incubation time were investigated separately. The optimal temperature and moisture level for α-galactosidase biosynthesis was found to be 30°C and 50%, respectively. The range of pH 5·5–6·5 was favourable. About 40–50 g of medium in 250-ml flask and inoculum over 1·0 × 106 spores were suitable for enzyme production. Seventy-five hours of incubation was enough for maximum α-galactosidase production. Substrate as wheat bran supplemented with soyabean meal and beet pulp markedly improved the enzyme yield in trays.Under optimum culture conditions, the α-galactosidase activity from Penicillium sp. MAFIC-6 indicated 185·2 U g−1 in tray of SSF.The process on α-galactosidase production in laboratory scale may have a potentiality of scaling-up.