Small conductance, calcium-activated potassium channels (SK channels) are present in most neurons, in denervated muscles and in several non-excitable cell types. In excitable cells SK channels play a fundamental role in the generation of the afterhyperpolarization which follows an action potential, thereby modulating neuronal firing and regulating excitability. To date, three channel subunits (SK1-3) have been cloned from mammalian brain. Since SK3 only has been shown to be expressed in muscles upon denervation, this channel may be involved in hyperexcitability and afterhyperpolarization observed in muscle cells in the absence of the nerve. Using confocal microscopy and SK3 specific antibodies, we demonstrate that SK3 immunoreactivity is present at the rat neuromuscular junction in denervated but also in innervated muscles. In denervated muscle fibers, SK3 is localized in the extrajunctional as well as the junctional plasma membrane, where it appears to be less abundant in the acetylcholine receptor-rich domains, corresponding to the crests of the postsynaptic folds. In innervated muscles, SK3 is not detectable in the muscle fiber but is present at the neuromuscular junction and seems to be localized presynaptically in the motor nerve terminals. Axonal accumulation of SK3 immunoreactivity occurs above and below a ligature of rat sciatic nerve, indicating that the SK3 protein is transported in both directions along the axons of the motor neurons. During rat development SK3 immunoreactivity is not found at the neuromuscular junction until day 35 of postnatal development when SK3 first appears in the motor neuron terminals. These results indicate that SK3 channels are components of the presynaptic compartment in the mature neuromuscular junction, where they may play an important regulatory role in synaptic transmission.