The isothiocyanate group was attached to the 4′-, 5′-, 6′-, or 7′-position of naltrindole in an effort to determine the importance of the position of this electrophilic group on the selectivity for subtypes of δ opioid receptors. All of the ligands were δ-selective when tested against standard agonists in smooth muscle preparations. However, the rank-order antagonism of δ antinociception in mice did not parallel the in vitro pharmacologic data. The 5′-isothiocyanate 2 was the most potent and selective antagonist in vivo, causing a 52-fold increase of the ED50 for [d-Ser2,d-Leu5]enkephalin-Thr6 (DSLET) and no increase for [d-Pen2,d-Pen5]enkephalin (DPDPE). The effect of each of the ligands on the binding of [3H]DSLET and [3H]DPDPE to guinea pig brain membranes clearly differentiated between the binding sites that recognize these radioligands. These studies provide additional evidence for the presence of two subtypes of δ opioid receptors.