Substrate specificity of the bacterial penicillinamidohydrolase (penicillinacylase, EC 3.5.1.11) from Escherichia coli was determined by measuring initial rates of enzyme hydrolysis of different substrates within zero order kinetics. Some N-phenylacetyl derivatives of amino acids and amides of phenylacetic acid and phenoxyacetic acid of different substituted amides of these acids or amides, structurally and chemically similar to these compounds, served as substrates. Significant differences in ratios of initial rates of the enzyme hydrolysis of different substrates were found using a toluenized suspension of bacterial cells or a crude enzyme preparation, in spite of the fact that the enzyme is localized between the cell wall and cytoplasmic membrane, in the so-called periplasmic space. N-phenylacetyl derivatives are the most rapidly hydrolyzed substrates. Beta-phenylpropionamide and 4-phenylbutyramide were not utilized as substrates. The substrate specificity of the enzyme is discussed with respect to a possible use of certain colourless compounds as substrates, hydrolysis of which yields chromophor products suitable for a simple and rapid assay of the enzyme activity.