Background: Virtual patients (VPs) have been recently integrated within different learning activities.Aim: To compare between the effect of using VPs in a collaborative learning activity and using VPs in an independent learning activity on students' knowledge acquisition, retention and transfer.Methods: For two different topics, respectively 82 and 76 dental students participated in teaching, learning and assessment sessions with VPs. Students from a female campus and from a male campus have been randomly assigned to condition (collaborative and independent), yielding four experimental groups. Each group received a lecture followed by a learning session using two VPs per topic. Students were administrated immediate and delayed written tests as well as transfer tests using two VPs to assess their knowledge in diagnosis and treatment.Results: For the treatment items of the immediate and delayed written tests, females outperformed males in the collaborative VP group but not in the independent VP group.Conclusion: On the female campus, the use of VPs in a collaborative learning activity is more effective than its use as an independent learning activity in enhancing students' knowledge acquisition and retention. However, the collaborative use of VPs by itself is not enough to produce consistent results across different groups of students and attention should be given to all the factors that would affect students' interaction.