The objectives of this study were to validate and compare the suitability of different primary cell culture systems as models to investigate peptide enzymatic stability following nasal administration. The degradation kinetics of a model peptide, leucine enkephalin (Tyr-Gly-Gly-Phe-Leu, Leu-Enk), was determined in four nasal cell culture systems: immersion, air-liquid interface, sequential monolayer-suspension, floating collagen. The influence of enzyme inhibitors (bestatin, puromycin) and Leu-Enk metabolite analogs (Tyr-Gly, Phe-Leu, Tyr-Gly-Gly, Gly-Phe-Leu) on the Leu-Enk degradation profile was also investigated. The disappearance of Leu-Enk in all the cell culture systems followed first order kinetics. The specific activity in the cell culture systems followed the rank: sequential monolayer-suspension (32.60 microM min(-1) mg(-1)) >air-liquid interface (15.19 microM min(-1) mg(-1)) >immersion (11.49 microM min(-1) mg(-1)) >floating collagen (4.57 microM min(-1) mg(-1)). At equimolar concentration, bestatin had a higher inhibitory effect than puromycin. The rate of hydrolysis of Leu-Enk was reduced significantly by co-incubation with Leu-Enk metabolite analogs. This study showed that immersion, sequential monolayer-suspension and air-liquid interface culture systems may be potentially suitable for further studies on peptide enzymatic stability following nasal administration.