Sympathetic neurons synthesize, transport, and release tissue-type plasminogen activators (t-PAs) and urinary-type plasminogen activators (u-PAs). We reported that t-PA enhances sympathetic neurotransmission and exacerbates reperfusion arrhythmias. We have now assessed the role of u-PA and plasminogen. Neurogenic contractile responses to electrical field stimulation (EFS) were determined in vasa deferentia (VD) from mice lacking t-PA (t-PA(-/-)), plasminogen activator inhibitor-1 (PAI-1(-/-)), plasminogen (plgn(-/-)), u-PA (u-PA(-/-)), and wild-type (WT) controls. Similar levels of t-PA were present in VD and cardiac synaptosomes of WT, PAI-1(-/-), plgn(-/-), and u-PA(-/-) mice, whereas t-PA was undetectable in t-PA(-/-) tissues. EFS responses were potentiated and attenuated in VD from PAI-1(-/-) and t-PA(-/-) mice, respectively, but indistinguishable from WT responses in VD from plgn(-/-) and u-PA(-/-) mice. Moreover, t-PA inhibition with t-PA(stop) decreased EFS response in WT mice, whereas u-PA(stop) did not. VD responses to ATP, norepinephrine, and K(+) in t-PA(-/-), PAI-1(-/-), plgn(-/-), and u-PA(-/-) mice were similar to those in WT, whereas t-PA(stop) did not modify VD responses to norepinephrine in WT, t-PA(-/-), and PAI-1(-/-) mice, indicating a prejunctional site of action for t-PA-induced potentiation of sympathetic neurotransmission. Indeed, K(+)-induced norepinephrine exocytosis from cardiac synaptosomes was potentiated in PAI-1(-/-), attenuated in t-PA(-/-) and not different from WT in u-PA(-/-) and plgn(-/-) mice. Likewise, ATP exocytosis was decreased in t-PA(-/-) and attenuated by t-PA(stop) in WT mice. Thus, t-PA-induced enhancement of sympathetic neurotransmission is a prejunctional event associated with increased transmitter exocytosis and independent of u-PA and plasminogen availability. This novel t-PA action may be a potential therapeutic target in hyperadrenergic states.