Native virions of human papillomaviruses (HPV) can be isolated from genital lesions only in very limited amounts. Recent studies have shown that virus-like particles can be obtained by expression of the capsid proteins using vaccinia virus recombinants or the baculovirus system. We now present the first detailed characterization of virus-like particles of a human papillomavirus associated with malignant genital lesions, HPV-33, produced in high yield using the baculovirus expression system. Assembly of the major capsid protein L1 alone or together with the minor capsid protein L2 has been obtained. Both spherical virus-like particles of 50-60 nm diameter and tubular structures of either 25-30 nm or 50-60 nm diameter and variable length were extracted from nuclei of the infected insect cells. However, predominantly 50- to 60-nm spherical particles were found in the cell culture medium in long-term infections. The particles have icosahedral symmetry and a density of 1.29 g/cm3 in cesium chloride corresponding to empty papillomavirus capsids. Immunoelectron microscopy confirmed the presence of L1 and L2 in the virus-like particles. The L2 protein seemed to be modified and was shown to be tightly associated with L1 using density gradient and sedimentation analysis. Approximately 50% of the L1 molecules are cross-linked by intermolecular disulfide bonds. This is the first example for the production of HPV-like particles containing both the major and the minor capsid protein using the baculovirus expression system.