Ram horn hydrolysate (RHH) was used as a substrate for the microbial production of citric acid using Aspergillus niger NRRL 330 in a submerged culture. Ram horns were hydrolyzed by treating with acid (6N-H2SO4) and neutralizing the solution. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHH were determined. The effect of different concentrations (1-10% v/v) of RHH on the production of biomass was investigated and a concentration of 8% RHH was found to be optimal. The content of citric acid in the culture broth containing 8% RHH (78 g/l) for 6 d was 26% higher than that of the control culture broth (62 g/l). From this result, RHH was demonstrated to be a suitable substrate for citric acid production. RHH may be a valuable supplement in biotechnology. (C) 2003 Elsevier Science Ltd. All rights reserved.