Here we report on the design and synthesis of the first nonpeptide, nonequilibrium δ opioid receptor antagonist, naltrindole 5'-isothiocyanate (1, NTII), that exhibits high pharmacologic selectivity both in vitro and in vivo. The design of NTII (1) was based on the attachment of the isothiocyanate group to naltrindole (2, NTI), a highly selective, reversible δ opioid receptor antagonist. Target compound 1 was prepared by reacting naltrexone with (p-nitrophenyl)hydrazine to form 5'-nitroindole 3, which was reduced to 5'-amino derivative 4 and then treated with thiophosgene (cyclization via Fischer indole synthesis). In vitro, NTII (100 nM) incubated with mouse vas deferens preparation (MVD) for 30 min irreversibly antagonized δ-selective agonists [D-Ala2,D-Leu5]enkephalin (DADLE) and [D-Pen2,D-Pen5]enkephalin (DPDPE) (no significant difference between pre- and postwash IC50 ratios), while showing no antagonism of μ-selective morphine or κ-selective ethylketazocine in guinea pig ileal longitudinal muscle preparation (GPI). Irreversible blockage was concentration-dependent (postwash IC50 ratios: 8.6 ± 0.5 at 100 nM vs 42.3 ± 5.8 at 500 nM for DADLE) and time-dependent (IC50 ratio 18 at 60 min). In vivo, icv administration of NTII (10 nmol) to mice 24 h prior to testing gave an ED50 ratio of 52.1 for δ-selective agonist DSLET, with ED50 ratios of 1.25 for morphine and 3.18 for κ agonist U-50,488H; antagonism peaked at 24 h and declined to 10 at 48 h. The in vitro and in vivo data demonstrate that NTII is a potent and selective, nonequilibrium δ opioid receptor antagonist, complementing existing δ-selective ligands with the added advantage of in vivo activity.