Adult Saccostrea glomerata were exposed to environmentally relevant concentrations of 4-nonylphenol (1 μg/L and 100 μg/L) and 17α-ethynylestradiol (5 ng/L and 50 ng/L) in seawater over 8 weeks. Exposures were performed to assess effects on vitellogenin induction and gonadal development during reproductive conditioning. Chronic direct estrogenicity within gonadal tissue was assessed via an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression assay (ER-CALUX®). Estradiol equivalents (EEQ) were greatest in the 100 μg/L 4-nonylphenol exposure (28.7 ± 2.3 ng/g tissue EEQ) while 17α-ethynylestradiol at concentrations of 50 ng/L were 2.2 ± 1.5 ng/g tissue EEQ. Results suggest 4-nonylphenol may be accumulated in tissue and is partly resistant to biotransformation; maintaining its potential for chronic estrogenic action, while 17α-ethynylestradiol, although exhibiting greater estrogenic potency on biological endpoints possibly exerts its estrogenic action before being rapidly metabolised and/or excreted. A novel methodology was developed to assess vitellogenin using high-performance liquid chromatography (HPLC). Exposure to both 17α-ethynylestradiol (50 ng/L) and 4-nonylphenol (100 μg/L) produced increases in vitellogenin for females, whereas males exhibited increases in vitellogenin when exposed to 50 ng/L 17α-ethynylestradiol only. Females exhibited greater vitellogenin responses than males at 50 ng/L 17α-ethynylestradiol only. Histological examination of gonads revealed a number of individuals exhibiting intersex (ovotestis) in 50 ng/L 17α-ethynylestradiol exposures. Male individuals in 1 μg/L and 100 μg/L 4-nonylphenol exposures and 5 ng/L 17α-ethynylestradiol were at earlier stages of spermatogenic development than corresponding controls.