Anatomists often use images in assessments and examinations. This study aims to investigate the influence of different types of images on item difficulty and item discrimination in written assessments. A total of 210 of 460 students volunteered for an extra assessment in a gross anatomy course. This assessment contained 39 test items grouped in seven themes. The answer format alternated per theme and was either a labeled image or an answer list, resulting in two versions containing both images and answer lists. Subjects were randomly assigned to one version. Answer formats were compared through item scores. Both examinations had similar overall difficulty and reliability. Two cross-sectional images resulted in greater item difficulty and item discrimination, compared to an answer list. A schematic image of fetal circulation led to decreased item difficulty and item discrimination. Three images showed variable effects. These results show that effects on assessment scores are dependent on the type of image used. Results from the two cross-sectional images suggest an extra ability is being tested. Data from a scheme of fetal circulation suggest a cueing effect. Variable effects from other images indicate that a context-dependent interaction takes place with the content of questions. The conclusion is that item difficulty and item discrimination can be affected when images are used instead of answer lists; thus, the use of images as a response format has potential implications for the validity of test items. Anat Sci Educ © 2012 American Association of Anatomists.