We have isolated and characterized alpha-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus. The peptide was classified as an alpha-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has homology to sequences of previously described alpha-conotoxins, particularly PnIA, PnIB, and ImI. However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry. Native EpI was shown to coelute with synthetic EpI. The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides. The activities of synthetic EpI and its unsulfated analogue [Tyr15]EpI were similar. Both peptides caused competitive inhibition of nicotine action on bovine adrenal chromaffin cells (neuronal nicotinic ACh receptors) but had no effect on the rat phrenic nerve-diaphragm (muscle nicotinic ACh receptors). Both EpI and [Tyr15]EpI partly inhibited acetylcholine-evoked currents in isolated parasympathetic neurons of rat intracardiac ganglia. These results indicate that EpI and [Tyr15]EpI selectively inhibit alpha3beta2 and alpha3 beta4 nicotinic acetylcholine receptors.