The goals of this study were to determine the steroid-binding specificity of the mouse salivary androgen-binding protein (ABP) family and to ascertain whether there might be other proteins in mouse saliva capable of binding steroids. The optimal conditions for testosterone binding by mouse salivary proteins were determined using a small-scale chromatography system to separate bound from unbound steroid. Testosterone binding appeared to be biphasic but was directly proportional to saliva concentration, with an optimum temperature of 37 °C in the second phase. These results were used to develop a steroid-binding protocol to study the steroid specificity of salivary proteins separated by electrophoresis. The ABP family bound testosterone and progesterone well and HO-progesterone and DHT to a lesser extent but did not bind either cholesterol or estradiol. Steroid structural comparisons suggest that binding by ABP is governed by the A ring of the steroid. Another protein that is not a member of the ABP family bound cholesterol specifically but no protein that specifically bound estradiol was observed.