Activities detectable in Streptococcus cremoris with the chymotrypsin substrate N-glutaryl-l-phenylalanine-4-nitroanilide and formerly designated endopeptidases P37 and P50 (F. A. Exterkate, Appl. Environ. Microbiol. 47:177-183, 1984) are both coupled peptidase reactions. These coupled reactions involve a membrane-bound, restricted l-alpha-glutamyl aminopeptidase which is responsible for the initial release of the glutaryl moiety. The subsequent reaction is catalyzed by either a so-called low-temperature or a high-temperature phenylalanyl aminopeptidase activity, both located at the outside surface of the membrane. Altered microenvironmental conditions created by the membrane-perturbing action of n-butanol or obtained by solubilization resulted in the removal of a restriction on the activity of l-alpha-glutamyl aminopeptidase and in a less efficient functioning of the coupled reactions; a long transient phase occurred before the steady state was reached. The results suggest that the in situ spatial organization is conducive to an efficient attuning of at least three peptidases which are located at the outer membrane surface and in the membrane. The possibility that peptidases in these locations exist as a cluster with physiological significance is discussed in relation to growth of S. cremoris in milk.