Cauliflower mosaic virus (CaMV) gene I encodes a protein (P1) that has been implicated in the control of virus movement in infected plants. To assist in the characterization of the mechanism of action of P1, gene I has been expressed efficiently in Spodoptera frugiperda (Sf) cells using recombinant baculovirus. Control of the expression of CaMV gene I by the polyhedrin late promoter in the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) resulted in very high levels of P1 accumulation late in the infection cycle. This was predominantly as insoluble inclusion bodies within the cytoplasm of infected Sf cells, and not extracellularly. Evidence from anomalous gel migration and sequence homology with an analogous viral protein (tobacco mosaic virus 30K) indicated that P1 may be post-translationally processed. However, neither phosphorylation nor glycosylation of P1 occurred in this system, suggesting a functional distinction between P1 and TMV 30K. P1 from insect cells and native P1 from infected plants were immunologically related, allowing the expressed product to be used in the preparation of anti-P1 serum for detecting P1 in plant extracts. The full-size (46 kD) P1 product from insect cells, from plants, and from in vitro translations of in vitro gene I transcripts all showed similar behavior on two-dimensional protein gels, with a major pI of 7.0. Using a combination of 4 M urea, 1 M NaCl, and high temperature, P1 was solubilized. Approximately 5% of the starting material remained in solution after dialysis and remained stable to freeze/thawing. This preparation should enable us to identify the biochemical function of P1 and to resolve its role in controlling virus spread.