OBJECTIVE: This study aimed to determine the impact of formulation (gel vs cream) and microneedle characteristics (length, number) on permeation of metronidazole through excised microneedle-treated skin. The long-term goal is to apply these results towards a pharmacokinetic study in human subjects with diverse skin types, using in vitro flux data to determine dosing conditions and ultimately establish in vitro-in vivo correlations. METHODS: Metronidazole release from 0.75% gel and cream was quantified with flow-through diffusion cells, using a cellulose membrane. Excised porcine skin was treated with stainless steel microneedles (500 or 800 μm length), to create 50 or 100 micropores. Metronidazole gel or cream was applied to microneedle-treated skin and replaced every 48 h for up to 7 days. Metronidazole permeation was quantified using HPLC. Intact skin (no microneedle treatment) served as controls. RESULTS: Metronidazole release was faster from the gel vs cream. At 7 days there was no difference between gel vs cream in total metronidazole permeated through intact skin. For both formulations, metronidazole permeation was significantly higher (vs intact skin) following microneedle application, regardless of microneedle length or micropore number. Increasing microneedle length and micropore number enhanced MTZ permeation multiple fold for both gel and cream. The greatest enhancement in total permeation for both formulations was achieved with the 800 μm MN, 100 micropore condition. CONCLUSIONS: Formulation and microneedle conditions both impacted metronidazole permeation. These data will be used to estimate in vivo serum concentrations after applying metronidazole to microneedle-treated skin in humans.