The purpose of this study was to investigate students' understanding about scientifically acceptable content knowledge by exploring the relationship between knowledge of diffusion and osmosis and the students' certainty in their content knowledge. Data was collected from a high school biology class with the Diffusion and Osmosis Diagnostic Test (DODT) and Certainty of Response (CRI) scale. All data was collected after completion of a unit of study on diffusion and osmosis. The results of the DODT were dichotomized into correct and incorrect answers, and CRI values were dichotomized into certain and uncertain. Values were used to construct a series of 2 X 2 contingency tables for each item on the DODT and corresponding CRI. High certainty in incorrect answers on the DODT indicated tenacious misconceptions about diffusion and osmosis concepts. Low certainty in incorrect or correct answers on the DODT indicated possible guessing; and, therefore no understanding, or confusion about their understanding. Chi-square analyses revealed that significantly more students had misconceptions than desired knowledge on content covering the Influence of Life Forces on Diffusion and Osmosis, Membranes, the Particulate and Random Nature of Matter, and the Processes of Diffusion and Osmosis. Most students were either guessing or had misconceptions about every item related to the concepts osmosis and tonicity. Osmosis and diffusion are important to understanding fundamental biology concepts, but the concept of tonicity should not be introduced to high school biology students until effective instructional approaches can be identified by researchers. (Contains 2 tables and 3 figures.)