The identity of the gonadotropin-releasing hormone (GnRH) form and the presence of GnRH-binding substances in the blood serum of the holocephalan, spotted ratfish ( Hydrolagus colliei), were investigated. The GnRH-like peptides in the serum were identified on the basis of relative hydrophobicity using reverse-phase HPLC. [His 5,Trp 7,Tyr 8]GnRH (chicken GnRH-II) was the only GnRH form detected in the serum. It has been previously shown to be the only GnRH form in the brain of this species. The presence of GnRH-binding substances was inferred by anomalous HPLC elution of GnRH, ultrafiltration behavior, and by the direct binding of iodinated GnRH analogues by blood serum components. The mean GnRH concentration in the extracted blood serum was 125 ± 11 pg ml −1 ( n = 5) in males and 64 ± 48 pg ml −1 ( n = 4) and 155 ± 26 ( n = 4) in two separate groups of females. Measurement of GnRH in the blood serum is complicated by the presence of GnRH-binding substances, which may cause the coprecipitation of GnRH during extraction with organic solvents. The high concentration of GnRH and the presence of GnRH-binding substances suggest that systemic blood is the route by which GnRH reaches the gonadotropes and/or that GnRH may have a hormonal role in H. colliei.