The discovery of large multiprotein complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant multiprotein production and multigene transfer applications.