Several neutralizing sites of the human papillomavirus (HPV) capsid are known to be critically dependent on the conformation of the capsid. However, efficient production of HPV16 capsids in mammalian cells has been difficult, possibly because the HPV genome contains negative regulatory elements. To circumvent these problems, we cloned the HPV16 L1 and L2 genes from a healthy HPV16-infected woman into a Semliki Forest virus based expression vector (P. Liljeström and H. Garoff, Biotechnology 9, 1356-1361, 1991). Recombinant HPV16 L1- or L2-producing Semliki Forest virus was generated and used for infection of mammalian cells. The HPV16 L1 and L2 proteins were efficiently expressed and the majority of the L1 protein self assembled into virus-like particles (VLPs). Coexpression of L1 and L2 resulted in incorporation of L2 into the VLPs. The particles had a density of approximately 1.3 g/ml as determined by density gradient centrifugation. Transmission electron microscopy revealed that the particles had a morphology similar to native virions. The HPV16 VLPs produced by the Semliki Forest virus expression system may be useful as a conformationally correctly assembled target for studies of HPV attachment, assembly, serology, or vaccination.