High mobility group protein 1 (HMG1) is an abundant non-histone chromosomal protein which plays a role in several nuclear events involving DNA. Here we demonstrate that HMG1 physically interacts with the human adeno-associated virus (AAV) Rep protein. HMG1 promotes the formation of Rep-DNA complexes and stimulates the activity of Rep in site- and strand-specific cleavage of DNA and the hydrolysis of ATP, functions required for viral gene regulation, replication and site-specific integration of viral DNA into human chromosome 19. We show that HMG1 enhances Rep-mediated repression of the AAV p5 promoter in transfected cells, suggesting that HMG1 and Rep also interact in vivo. HMG1, Rep and DNA can be immunoprecipitated as a ternary complex. Kinetic studies indicate that complexes of Rep with DNA have similar stabilities in the presence and absence of HMG1.These results suggest that the effect of HMG1 on Rep binding is exerted at the step of complex formation and thereby may reflect an activity of HMG1 in promoting the assembly of complex cellular nucleoprotein structures.