Succinyl trialanine p-nitroanilide [Suc-(Ala)3-pNA] hydrolytic activity, an enzymatic activity related to elastase, in vascular wall was detected and partially characterized. Subcellular distribution of this activity closely paralleled that of plasma membrane marker enzymes, 5'-nucleotidase, and phosphodiesterase I, suggesting its association with the vascular muscle plasma membranes. The same distribution of elastolytic activity was observed. Hydrolytic activity toward Suc-(Ala)3-pNA was inhibited by EDTA, ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid but not by trypsin inhibitor. Enzyme activities were different not only between aortic muscle extracts from young and mature rats, but also among the extracts from elastic and muscular arteries, specific activity being higher in the aortas of young animals or in elastic arteries, respectively. The activity studied [Suc-(Ala)3-pNAase] in vascular wall may play a role in vascular connective tissue metabolism as well as function.