A 300-bp deletion was made in gene I of a multimeric copy of cauliflower mosaic virus (CaMV). The deleted and wild-type constructs were mobilized into Agrobacterium tumefaciens and used for the inoculation of plants and leaf discs of the host plant, turnip, by ’agroinfection.’ Infection induced by the wild-type construct gave characteristic viral symptoms on the inoculated and systemically infected leaves of whole plants whereas no infection was detected using the deletion mutant. The possibility that the deletion in gene I affected a cis-related function (e.g., encapsidation) was tested by complementation of mutant gene I with wild-type CaMV. Double infection experiments showed that replication and spread of both viruses occurred without detectable recombination of the mutant. In contrast to the situation in plants, agroinfection (Grimsley et al., 1986), Proc. Natl. Acad. Sci. USA 83, 3282-3286) of leaf discs, detected as the accumulation of encapsidated forms of virion DNA, was recorded for both the wild-type and the deleted constructs. The reduced level of virus accumulation from the mutant was consistent with virus replication occurring only in cells involved in the primary interaction with Agrobacterium, and accordingly progeny viral DNA was only detected in cells around the edge of the tissue disc. These data provide definitive evidence that CaMV gene I encodes a movement protein and show that this function can act in trans in a virus infection. © 1993 Academic Press, Inc.