The bacteriophage G4 origin of complementary strand DNA synthesis, G4 ori, contains several regions of potential secondary structure. In this study, we ask whether DNA secondary structure is important for G4 ori function in vivo. Point mutations were generated within a region of potential secondary structure so as to disrupt intrastrand base pairing. These mutations led to a strong temperature-dependent reduction in ori function in vivo. A double point mutation which introduces the same base substitutions without destabilizing intrastrand base pairing did not cause a temperature-dependent disruption in ori function. The double mutant did display a slight temperature-independent reduction in ori function compared to the wild-type G4 ori. Based on these findings, we conclude that DNA secondary structure, as well as recognition of specific sequences, is required for G4 ori activity in vivo.