Screening of a representative series of immobilized penicillin G acylase biocatalysts (enzyme, cells) using enzyme flow microcalorimetry is described. Immobilized penicillin G acylase biocatalysts were either prepared in the laboratory by various techniques or obtained from four commercial manufacturers. An industrial strain of Escherichia coli was entrapped in (poly)acrylamide gel or hardened calcium pectate gel. Semi-purified enzyme was immobilized in various ways-either by covalent binding to oxirane-acrylic beads or chlorotriazine bead cellulose or by entrapment in (poly)acrylamide gel. The validity of the enzyme flow microcalorimetry results was corroborated by a pi-I-stat method, showing enzyme flow microcalorimetry to be a suitable method for rapid screening of immobilized biocatalysts regardless of the immobilization technique, carrier type or the biocatalyst source.