The omega-conotoxins are small, disulfide-rich peptides which inhibit voltage-sensitive calcium channels. Biotinylated omega-conotoxins are potentially useful reagents for characterizing distinct subsets of calcium channels. We describe the preparation and characterization of biotinylated derivatives of two specific omega-conotoxins, GVIA and MVIID, which bind different calcium channel subtypes. Eight biotinylated derivatives were tested; all specifically displaced binding of the radiolabeled unbiotinylated omega-conotoxin. In general, the addition of one biotin moiety decreased the apparent affinity for the receptor target site by only approximately 10-fold. However, derivatization of omega-conotoxin MVIID at the Lys10 residue caused a much more marked effect, a ca 500-fold decrease in affinity. These results indicate that the vicinity of the Lys10 residue of omega-conotoxin MVIID may be more critical for binding to the receptor target site than regions around other amino groups in omega-conotoxins GVIA and MVIID. Thus, high affinity biotinylated omega-conotoxin GVIA and MVIID derivatives have been chemically defined; the biotin groups have been shown to be accessible to streptavidin. Given the commercial availability of streptavidin coupled to various reporter groups, the biotinylated omega-conotoxin derivatives described here should be widely useful for fluorescence, electron microscopic or immunological applications.