Background and purpose:  The aim of this study was to assess the relative bioavailability of diazepam after administration of diazepam itself or as a water-soluble prodrug, avizafone, in humans. Experimental approach:  The study was conducted in an open, randomized, single-dose, three-way, cross-over design. Each subject received intramuscular injections of avizafone (20 mg), diazepam (11.3 mg) or avizafone (20 mg) combined with atropine (2 mg) and pralidoxime (350 mg) using a bi-compartmental auto-injector (AIBC). Plasma concentrations of diazepam were quantified using a validated LC/MS–MS assay, and were analysed by both a non-compartmental approach and by compartmental modelling. Key results:  The maximum concentration (Cmax) of diazepam after avizafone injection was higher than that obtained after injection of diazepam itself (231 vs. 148 ng·mL−1), while area under the curve (AUC) values were equal. Diazepam concentrations reached their maximal value faster after injection of avizafone. Injection of avizafone with atropine–pralidoxime (AIBC) had no effect on diazepam Cmax and AUC, but the time to Cmax was increased, relative to avizafone injected alone. According to the Akaike criterion, the pharmacokinetics of diazepam after injection as a prodrug was best described as a two-compartment with zero-order absorption model. When atropine and pralidoxime were injected with avizafone, the best pharmacokinetic model was a two-compartment with a first-order absorption model. Conclusion and implications:  Diazepam had a faster entry to the general circulation and achieved higher Cmax after injection of prodrug than after the parent drug. Administration of avizafone in combination with atropine and pralidoxime by AIBC had no significant effect on diazepam AUC and Cmax.