Spermatogenesis involves interactions between the seminiferous tubules, which contain developing germ cells and somatic Sertoli cells, and the interstitial space which contains steroidogenic Leydig cells, macrophages and blood vessels. Because little is known about soluble factors that potentially participate in intercellular communication between the various cell types, our aim was to investigate changes in the proteome of testicular interstitial fluid (TIF) that surrounds the seminiferous tubules following selective germ cell damage induced by heat treatment. Adult (70 days old) Sprague-Dawley rats were anaesthetised and their scrota immersed in a water bath at 43°C for 15 minutes. This treatment results in the loss of pachytene spermatocytes (PSC) and round spermatids (rST) from the epithelium with maximum damage 2-6 weeks later, but spermatogenesis recovers by 10-12 weeks(1). TIF was collected from treated and age-matched untreated control rats (n=4/group) at 1, 2, 4, 8, 12, and 14 weeks following heat treatment. After removal of abundant proteins, TIF proteins were identified by LC-MS/MS with statistically different candidates selected by ANOVA. The potential cellular origin of TIF proteins was assessed using datasets from a microarray of isolated germ and Sertoli cells(2) and an RNASeq of mouse testes with germ- +/- Sertoli cell ablation(3). Overall, 1729 unique proteins were identified in TIF, with 31 proteins significantly (p<0.05) suppressed 2-22 fold at the 8 week maximal damage timepoint. These proteins included potential markers of Sertoli cells (n=3), PSC and rST (n=7), spermatogonial (n=1) and interstitial cells (n=1). This group included several germ cell heat shock-related proteins, along with ubiquitin-conjugating and calcium regulating proteins. Interestingly, this strategy also revealed various Sertoli and spermatogonial proteins altered in the 1 week group, likely indicative of an early response to heat by the epithelium. This proteomic approach opens a new direction to study intercellular communication in the testis.