Assembly and budding formation of Gag precursor is thought to be important for particle formation in retrovirus. To investigate the role of Gag precursor proteins, Pr53gag, of the HTLV-II (Human T-cell Leukemia Virus type II), we induced the expression of unprocessed Pr53gag in recombinant baculovirus-infected insect cells. The protein was assembled and immature empty particles were released from the insect cells, which were ascertained by Western blotting, sucrose density gradients analysis and electron microscopy. When the Gag-Pro-Pol precursor proteins were expressed by the recombinant baculovirus, mature particles were produced. However, deletion of the pol gene did not affect the viral formation, which was different from the process of virion production of HIV-1. This was explained by the facts that HTLV-II could yield Gag-Pro precursor protein, which was expected to have protease activity by itself. Moreover, the results were consistent with low production of Gag-Pro-Pol precursor proteins, which were translated by double frameshifting.