Histology stands as a major discipline in the life science curricula, and the practice of teaching it is based on theoretical didactic strategies along with practical training. Traditionally, students achieve practical competence in this subject by learning optical microscopy. Today, students can use newer information and communication technologies in the study of digital microscopic images. A virtual microscopy program was recently introduced at Ghent University. Since little empirical evidence is available concerning the impact of virtual microscopy (VM) versus optical microscopy (OM) on the acquisition of histology knowledge, this study was set up in the Faculty of Medicine and Health Sciences. A pretest-post test and cross-over design was adopted. In the first phase, the experiment yielded two groups in a total population of 199 students, Group 1 performing the practical sessions with OM versus Group 2 performing the same sessions with VM. In the second phase, the research subjects switched conditions. The prior knowledge level of all research subjects was assessed with a pretest. Knowledge acquisition was measured with a post test after each phase (T1 and T2). Analysis of covariance was carried out to study the differential gain in knowledge at T1 and T2, considering the possible differences in prior knowledge at the start of the study. The results pointed to non-significant differences at T1 and at T2. This supports the assumption that the acquisition of the histology knowledge is independent of the microscopy representation mode (VM versus OM) of the learning material. The conclusion that VM is equivalent to OM offers new directions in view of ongoing innovations in medical education technology. Anat Sci Educ 6: 307–315. © 2013 American Association of Anatomists.