A spectrophotometric method to determine gallic acid, residual gallotannin and tannin acyl hydrolase (EC 3.1.1.20) activity during microbial hydrolysis of pentagalloyl glucose is described. The following equations have been developed to estimate gallotannin and gallic acid in the incubation medium by absorbance measurements at two different wavelengths: concentration of gallotannin (microg ml(-1))=34.41 (A293.8)-6.98 (A254.6); concentration of gallic acid (microg ml(-1))=21.77 (A254.6)-17.17 (A293.8). As compared to Aspergillus and Penicillium, the fungal genera extensively studied for the production of this enzyme, Fusarium solanii and Trichoderma viride exhibited higher enzyme activity showing approximately 88 and 84 mole percent conversion respectively after a 24 h incubation period.