The cotton fiber is a model system to study cell wall biosynthesis because the fiber cell elongates (∼3 cm in ∼20 days) without mitosis. In this study, developing cotton ovules, examined from 1 day before anthesis (DBA) to 2 days post-anthesis (DPA), that would be difficult to investigate via classical carbohydrate biochemistry were probed using a battery of antibodies that recognize a large number of different wall components. In addition, ovules from these same stages were investigated in three fiberless lines. Most antibodies reacted with at least some component of the ovule, and several of the antibodies reacted specifically with the epidermal layer of cells that may give clues as to the nature of the development of the fibers and the neighboring, nonfiber atrichoblasts. Arabinogalactan proteins (AGPs) labeled the epidermal layers more strongly than other ovular tissue, even at 1 DBA. One of the AGP antibodies, CCRC-M7, which recognizes a 1➔6 galactan epitope of AGPs, is lost from the fiber cells by 2 DPA, although labeling in the atrichoblasts remained strong. In contrast, LM5 that recognizes a 1➔4 galactan RGI side chain is unreactive with sections until the fibers are produced and only the fibers are reactive. Dramatic changes also occur in the homogalacturonans (HGs). JIM5, which recognizes highly de-esterified HGs, only weakly labels epidermal cells of 1 DBA and 0 DPA ovules, but labeling increases in fibers cells, where a pectinaceous sheath is produced around the fiber cell and stronger reaction in the internal and external walls of the atrichoblast. In contrast, JIM7-reactive, highly esterifed HGs are present at high levels in the epidermal cells throughout development. Fiberless lines displayed similar patterns of labeling to the fibered lines, except that all of the cells had the labeling pattern of atrichoblasts. That is, CCRC-M7 labeled all cells of the fiberless lines, and LM5 labeled no cells at 2 DPA. These data indicate that a number of polysaccharides are unique in quantity or presence in the epidermal cell layers, and some of these might be critical participants in the early stages of initiation and elongation of cotton fibers.