One of the neuropsychological deficits that can result from a stroke is the neglect phenomenon. Neglect has traditionally been assessed with paper-and-pencil tasks, which are administered within the reaching space of a person. The purpose of this explorative study is to investigate whether it is possible to assess neglect in the extrapersonal space based on the performance of acute stroke patients, chronic stroke patients and healthy elderly in orientation and exploration tasks when immersed in a three-dimensional (3D)- virtual environment. Six able-bodied healthy elderly and 12 stroke patients (six subacute and six chronic) participated in this explorative cross-sectional study. Neglect was assessed by means of the 3D neglect test. Different parameters were measured at two challenge levels with increasing difficulty. In the easiest level significant differences between the groups were found for total time spent in the test, mean response time left field of vision, and mean response time in the left field of vision of the left virtual reality environment. Differences in search patterns showed that subacute stroke patients had a much more fuzzy search pattern in scanning the environment than healthy elderly and chronic stroke patients. With respect to the more difficult level results showed significant differences between healthy elderly and the total group of stroke patients. The results of this study suggest that a 3D neglect test by means of virtual reality has the potential to detect and measure unilateral neglect.