Spinach (Spinacia oleracea) seed from a commercial breeding line suspected of harboring cucumber mosaic virus (CMV) was analyzed for seed transmission of the virus. Initial seed grow-out tests and enzymelinked immunosorbent assay studies indicated that CMV was present in this seed lot at a level of nearly 15%. To verify these results and gain insight into the mechanism of seed transmission, four combinations of crosses between healthy and/or infected parent plants were conducted. None of the spinach seedlings derived from crossing healthy male and healthy female plants contained CMV, whereas a portion of seedlings derived from all of the other three crosses, i.e., healthy male and infected female, infected male and healthy female, and infected male and infected female plants, were infected with CMV. The results demonstrate that CMV is seed transmitted in spinach and indicate that both male and female parent plants can serve as infection sources. Ultrastructural studies, including immunogold labeling, revealed the presence of virus particles in the cytoplasm of ovary wall cells, ovule integuments and nucellus, anther, and seed-coat cells, as well as fine fibril-containing vesicles and electron-dense inclusions of amorphous aggregates in the central vacuoles of these cells. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 860-bp cDNA fragments containing the CMV coat protein (CP) gene from the embryo, endosperm, and pollen tissues of CMV-infected plants. Taken together, these studies indicate that CMV occurs in virtually all spinach reproductive tissues. Analysis of several RT-PCR amplified and cloned CP genes and flanking sequences from parent and progeny plants revealed that the spinachinfecting CMV was a member of subgroup II. Furthermore, cDNA sequencing and restriction endonuclease mapping consistently revealed two sequence variants, designated SP103 and SP104, in most plants analyzed. These data suggest that there may have been mixed infections of two distinct, seed-transmitted CMV variants in spinach.