The critical parameters, such as slant age, mycelium age and contact time of mycelium with lytic enzymes which affects the formation of protoplast from the Penicillium griseofulvum mycelium were studied using a novel technique called response surface methodology. The design used to optimize these parameters was full factorial central composite design. The number of protoplasts, viability of protoplasts and griseofulvin yield are the three responses studied in this communication. The individual optimum values of slant age, inoculum age and contact time obtained are 187.1, 30.81, 21.4 h, respectively, for number of protoplast/ml 68.92, 33.01, 9.88 h, respectively, for percentage viability of protoplasts and 230.2, 73.6, 31.2 h, respectively, for griseofulvin. In order to obtain a single optimum value for all the three responses, a multi-response analysis was carried out using a generalized distance approach. The simultaneous optimum values of slant age, inoculum age and contact time are 133.78, 37.1 and 15.97 h, respectively, for all the three responses. © 2003 Elsevier Science B.V. All rights reserved.