Aminopeptidase I is a membrane-bound metalloenzyme isolated from B. stearothermophilus which is thermostable and requires Co2+ for activity. The Co, Zn, and metal-free enzyme were titrated with ANS, and cooperative binding was noted with the active Co enzyme but not with the Zn (5% active) or apo (inactive) enzymes. There are a number of sites for ANS on each of the three enzyme forms and the agreement between the association constants of the Zn enzyme (identical and independent sites) and the non-cooperative sites of the Co enzyme suggest that these sites are intrinsically similar. However, binding to the first of these sites in the Co enzyme triggers a cooperative binding of a second molecule of ANS, and this cooperative binding is related to a concomitant decrease in enzymatic activity. The correspondence can be shown by comparison of the inhibition constant for the hydrolysis of Gly-Leu-Tyr (Ki-1=13,300 cm3/mmol) and the association constant for the cooperating site (12,500 cm3/mmol). The significance of these observations is discussed in terms of the nature of the binding sites and the possible consequences of the interactions on the regulation of aminopeptidase I activity.