The complete nucleotide sequence of RNA 3 of a Spanish isolate of cucumber mosaic virus (CMV-24) has been determined. The encoded putative cell-to-cell movement protein (3a protein) and the coat protein are 279 and 218 amino acids long, respectively. The 3a protein was expressed in Escherichia coli using the vector pT7-7 and was used to raise an immunoserum. We have followed the time course of accumulation of the 3a protein, in parallel to that of the coat protein, and its subcellular localization as a function of time after CMV-24 infection on tobacco plants. The maximum accumulation level of the 3a protein was reached at early stages of infection, being detected in the cytosolic and the cell wall fractions. At later stages of infection, a decline in accumulation levels of the 3a protein was observed, and the protein was essentially associated with the cell wall fractions. These data were corroborated by immunocytochemistry performed in both infected and 3a-expressing transgenic tobacco plants.