The production of the fungus Agaricus bisporus ATTC 10893 was studied in submerged fermentation with horn hydrolysate as the main substrate source. First, horns were ground and 35 g of the horn powder were chemically hydrolyzed (acid hydrolysis). As a result of this process, 30 of the 35 g horn powder (85.7%) could be hydrolyzed. Hydrolyzed material was completed to 400 ml with deionized water and this resulting solution was termed as crude horn hydrolysate (CHH). The contents of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of CHH were determined. It was found that it has both organic and inorganic materials sufficient for use as a substrate in the production of Agaricus bisporus mycelium. The CHH was enriched by addition of glucose and KH 2PO 4. The effects of different concentrations (1–7% v/v) of CHH on the growth of A. bisporus were investigated and 2% of the CHH (Horn Broth=HB) was found to be optimal. The HB contains 20 g of glucose, 20 ml of CHH and 1 g of KH 2PO 4 per liter of laboratory quality water. The optimum growth conditions for the A. bisporus in HB were as follows: Initial pH: 6; temperature: 26 °C; fermentation time: 8 days and agitation: 150 rpm. Under these optimal conditions, initial carbohydrate content of HB was reduced from 2.02 to 0.2% and the biomass yield was 10.8 g l −1. The biomass contained about 47.1% of protein, 5.8% of fat and 7.9% of ash (on a dry-weight basis). Amino acid content of biomass was compared with both the FAO standard and fruiting body. The biomass contained all of the essential amino acids but the amounts of the essential amino acids were somewhat lower than that of the FAO standard, while it was comparable with that of fruiting body. The results with HB were also compared with previously reported data on edible mushroom mycelium grown on some substrates. It was found that HB could be used as a substrate for the production of Agaricus bisporus mycelium.