B-50 (GAP-43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin-binding domain of B-50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B-50 into streptolysin O-permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N-terminal residues 39-43 of rat B-50 dose-dependently inhibited Ca(2+)-induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C-terminal-directed (residues 132-213) anti-B-50 antibodies were without effect in the same dose range. NM2 inhibited PKC-mediated B-50 phosphorylation at Ser41 in synaptosomal plasma membranes and permeated synaptosomes, inhibited 32P-B-50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca2+. Similar concentrations of NM6 did not affect B-50 phosphorylation or dephosphorylation or B-50/calmodulin binding. We conclude that the N-terminal residues 39-43 of the rat B-50 protein play an important role in the process of Ca(2+)-induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca(2+)- and phosphorylation-dependent fashion.