In this study, we describe the sequence for the fathead minnow (Pimephales promelas) vitellogenin 3 gene (vg3) and compare the responses of vg1 and vg3 following exposure to steroidal estrogens and androgens. The fathead minnow vg3 sequence is the second nucleotide sequence described in teleosts, following the original description of this isoform in zebrafish (Danio rerio). Following a brief water exposure (24h) to 2, 5, and 10 ng/L 17alpha-ethynylestradiol (EE2), both vg1and vg3 were upregulated in male liver. However, levels of vg3 induction were four orders of magnitude lower than levels of induction of vg1. Suppression of vg in female liver following exposure to 50 or 500 ng/L of the synthetic androgen 17beta-trenbolone occurs at similar magnitude for both vg1 and vg3 isoforms. The results of this study support the use of vg1 as an indicator of estrogenic exposure in male fish and indicate the potential for vg1 and /or vg3 for use as indicators of androgenic exposure.