We present data indicating that a host protein is important for function of HIV-1 preintegration complexes (PICs) in vitro. PICs partially purified from infected cells were subjected to gel filtration in 600 mM KCl, which removed a factor required for integration without fully disrupting PICs. Addition of an extract from uninfected cells restored activity. Fractionation of the complementing activity yielded HMG I(Y), a nonhistone chromosomal protein important for transcriptional control and chromosomal architecture. Complementing activity could be isolated from PICs, and activity could be depleted from such fractions with an antibody against HMG I(Y). Recombinant HMG I(Y) also complemented salt-stripped complexes. The finding that a host protein is required for integration by HIV PICs parallels findings in several well-studied transposition and site-specific recombination systems.