Originally the macrophage migration inhibitory factor (MIF) was described as a classical T-cell cytokine. Recently, a much broader tissue distribution for MIF has been revealed. We demonstrated that MIF protein and mRNA are present in the Leydig cells of the normal adult rat testis. Addition of recombinant MIF to cultures of rat seminiferous tubules resulted in decreased secretion of inhibin, whereas follistatin and activin levels remained unchanged, suggesting a paracrine role for MIF in Sertoli cell regulation. Furthermore, MIF showed unique compensatory production in the rat testis. Depletion of the original MIF source, the Leydig cells, by the specific toxin EDS prompted MIF expression by the previously negative Sertoli cells. Leydig cell re-population of the interstitial tissue by precursor cells resulted in a switch back to production by Leydig cells. Therefore, testicular MIF appears to be under very tight paracrine control. MIF has thus been identified as a new mediator in the cross-talk between Leydig cells and the somatic cells of the seminiferous tubules of the rat testis.