Abstract: This paper presents a method and results of intrinsic kinetic parameters for hydrolysis of 7-phenylacetamide-aminodesacetoxycephalosporanic acid catalyzed by fibrous immobilized penicillin amidase. The initial velocities were determined under intrafibrous and external diffusion limitations. The apparent kinetic parameters were obtained either by Lineweaver-Burk plot or by Dixon plots. The intrinsic parameters were obtained by reploting the corresponding apparent values against the superficial substrate feeding velocities. The intrinsic K_m, K_p and K_q were found to be 6.9, 16.8 and 94.4 mmol/L, respectively. By comparing with the data reported for the catalytic hydrolysis of benzylpenicillin catalyzed by the same immobilized enzyme, we suggested that the method just reported can be used for kinetic parameter determination in the presence of intrafibrous and external diffusion limitations.