Biology student mastery regarding the mechanisms of diffusion and osmosis is difficult to achieve. To monitor comprehension of these processes among students at a large public university, we developed and validated an 18-item Osmosis and Diffusion Conceptual Assessment (ODCA). This assessment includes two-tiered items, some adopted or modified from the previously published Diffusion and Osmosis Diagnostic Test (DODT) and some newly developed items. The ODCA, a validated instrument containing fewer items than the DODT and emphasizing different content areas within the realm of osmosis and diffusion, better aligns with our curriculum. Creation of the ODCA involved removal of six DODT item pairs, modification of another six DODT item pairs, and development of three new item pairs addressing basic osmosis and diffusion concepts. Responses to ODCA items testing the same concepts as the DODT were remarkably similar to responses to the DODT collected from students 15 yr earlier, suggesting that student mastery regarding the mechanisms of diffusion and osmosis remains elusive. (Contains 7 tables and 2 figures.)