Stac protein (named for its SH3 and cysteine rich domains) was first identified in brain 20 years ago, and is currently known to have three isoforms. Stac2, Stac1 and Stac3 transcripts are found at high, modest and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca(2+) channels. Over-expression of the three Stac isoforms eliminated Ca(2+)-dependent inactivation (CDI) of L-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-L-type current. Using heterologous expression in tsA201 cells (together with beta and alpha2-delta1 auxiliary subunits) we found that CDI for CaV1.2 and CaV1.3 (the predominant, neuronal L-type Ca(2+) channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, CaV2.1, was not. For CaV1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel's C-terminus. Within the Stac proteins, a weakly conserved segment, containing approximately 100 residues and linking the structurally conserved PKC C1 and SH3_1 domains, was sufficient to fully suppress CDI. The presence of CDI for L-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons, and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared to neonate. Taken together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca(2+) entry via neuronal L-type channels.SIGNIFICANCE STATEMENTStac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal muscle L-type Ca(2+) channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here we report that one likely function of neuronal Stac proteins is tuning Ca(2+) entry via L-type, but not that via non-L-type, Ca(2+) channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation of L-type channel Ca(2+) signaling in these brain regions.