Temperature-related sequence 4 (Trs4) has been identified as a testis-specific gene with expression sensitive to the abdominal temperature changes induced by artificial cryptorchidism. In murine testes, Trs4 mRNA was detected in round spermatids and its protein was localized mainly in the elongating spermatids as well as in the acrosomes and tails of mature spermatozoa. Using a yeast two-hybrid screening system, we identified Rshl-2, Gstmu1, and Ddc8 as putative binding partners of the Trs4 protein in mouse testes. Their interactions were confirmed by in vivo and in vitro binding assays. Further studies demonstrated that Ddc8, a newly identified gene with unknown functions, displayed a similar expression pattern with Trs4 in mouse testes. In particular, Trs4, Ddc8, and Rshl-2 proteins were co-localized to the tails of mature spermatozoa. These results suggested that Trs4 might be involved in diverse processes of spermiogenesis and/or fertilization through interactions with its multiple binding partners. (C) 2009 Elsevier Inc. All rights reserved.