Phospholipid-binding proteins in the male genital tract are characterized by differing numbers Fn-2 modules (B-domain) carrying N-terminal extensions (A-domain) of variable length. In the stallion, three different proteins were identified, SP-1, SP-2, and EQ-12. SP-1 and SP-2 of the AA'BB'- and ABB'-type, respectively, are major proteins of the seminal plasma. Here we report the cDNA sequences of SP-1, and of a new member of the SP-2 family (SPnew) and the partial characterization of their iso- and glycoforms. The phosphorylcholine (PC)-binding ability of the long Fn-2 protein, EQ-12, with four tandemly arranged Fn-2 modules was determined by PC-affinity chromatography. Expression patterns of EQ-12, and the SP-proteins were studied by means of RT-PCR, Northern blot analysis and immunological approaches indicating differential expression along the male reproductive tract. The vast majority of the short SP-1 and SP-2 proteins are produced by the ampulla whereas EQ-12 originates from the epididymis. Indirect immunofluorescence microscopy of sperm isolated from different regions of the epididymis and Western blot analysis indicate that both, the long and the short Fn-2 proteins associate to the sperm surface during post-testicular maturation. Sperm binding of Fn-2 proteins at the post-acrosome and midpiece was at first detected in the corpus epididymis. Enhanced fluorescence intensity after ejaculation point to an increased number of molecules bound to the sperm surface. The function of these proteins is discussed in regard to their structure–function relationships. Mol. Reprod. Dev. 70: 45–57, 2004. © 2004 Wiley-Liss, Inc.