To investigate whether items of the Medical Outcomes Study (MOS) 36-Item Short-Form Health Status Survey (SF-36) exhibited differential item functioning (DIF) with respect to age, education, race, and gender. The data for this study come from two large national datasets, the MOS and the 1990 National Survey of Functional Health Status (NSFHS). We used logistic regression to identify items exhibiting DIF. We found DIF to be most problematic for age comparisons. Items flagged for age DIF were vigorous activities, bend/kneel/stoop, bathing or dressing, limited in kind of work, health in general, get sick easier than others, expect health to get worse, felt calm and peaceful, and all four vitality items. Items flagged for education DIF include vigorous activities, health in general, health is excellent, felt calm and peaceful, and been a happy person. Vigorous activities, walk more than a mile, health in general, and expect health to get worse were identified as DIF when comparing African-Americans with whites. No items were identified for gender DIF. We found several consistent patterns of DIF using two national datasets with different population characteristics. In the current study, the effect of DIF rarely transferred to the scale level. Further research is needed to corroborate these results and determine qualitatively why DIF may occur for these specific items.