t-PA has a widespread neuroendocrine distribution including prominent expression in chromaffin cells of the sympathoadrenal system. Chromaffin cell t-PA is sorted into catecholamine storage vesicles and co-released with catecholamines in response to sympathoadrenal activation, suggesting that catecholamine storage vesicles may serve as a reservoir for the rapid release of t-PA. Chromogranin A (CgA), a major core protein in secretory vesicles throughout the neuroendocrine system, may play a crucial role in targeting proteins into the regulated secretory pathway, by forming aggregated "granin" complexes to which other proteins destined for the regulated secretory vesicle bind and become separated from constitutively secreted proteins in the trans-Golgi network (TGN). Formation of such complexes is facilitated by conditions of the TGN (low pH, high Ca+2). We tested the hypothesis that t-PA interacts specifically with CgA and that this interaction is enhanced under conditions of the TGN. Immobilized t-PA was incubated with 125I-CgA. t-PA interacted specifically and saturably with CgA and the interaction was domain-specific, mediated by the EGF/finger and kringle 1 domains of t-PA and by a specific internal hydrophilic domain within CgA (KERTHQQKKHSSYEDELSEVL) as assessed by antibody and peptide competition studies. The interaction of t-PA with aggregated CgA complexes may play a role in the targeting of t-PA and its release from neurosecretory cells. These results may have broad implications for the regulation of local neurosecretory cell plasminogen activation under both normal physiological conditions and pathological conditions including cerebral ischemia.