Study of hormonal regulation of fruit development requires quantitative measurement of reproductive tissues at different developmental stages. This study reports the application of ELISA (enzyme-linked immunosorbent assay) for the measurement of free indole-3-acetic acid (IAA), dihydrozeatin riboside (DHZR) and abscisic acid (ABA) in ovules of peanut (Arachis hypogaea L., cv. NC 6). IAA levels in apical and basal ovules gradually increased with aerial peg elongation from 4 to 10 d after pollination (DAP), but decreased when peg elongation ceased by 14 DAP. Higher DHZR levels were observed in aerial ovules at 10 DAP, and the ABA level was highest at 1 DAP. Aerial apical ovules contained higher IAA and DHZR levels than basal ovules. At day 1 after soil penetration (DASP), IAA and ABA levels in both ovules decreased, whereas DHZR levels increased. Peg tip swelling and ovary enlargement at 5 DASP were accompanied by increased levels of all three hormones. The highest levels of the hormones were observed in both ovules at 7 DASP, i.e., when embryos were at the globular stage and ovaries were rapidly enlarging to form pods. Basal ovules also had higher hormonal levels than apical ovules after soil penetration. Progressive changes in hormone levels during peanut ovule and embryo development suggest a physiological relationship between the two events.