Abstract: Testosterone is the main androgen in the male, mainly synthesized by the Leydig cells in the interstitial tissues of the testes. It is a pro-hormone for biologically active 5-areduced androgen and estrogens and is produced by the action of luiteinizing hormone (LH) through cyclic adenosine monophosphate (cAMP) pathway. This classical pathway of steroidogenesis is a major but not sole player in testosterone synthesis in the Leydig cells. Other intracellular signaling systems also play important role in Leydig cells via which several endogenous bio-chemicals have been found to actively participate either in stimulation or inhibition of testosterone production. One of the factors that have been poorly investigated for their autocrine and paracrine action in the testosterone production by the Ledyig cells are the neurotransmitters. In this experiment we investigate some putative neurotransmitters (NTs) namely dopamine (DA), 3, 4-dihydrophenylacetic acid (DOPAC), epinephrine (E), norepinephrine (NE), 5-hydroxytryptamine (5-HT), homovanillic acid (HVA), 3-methoxytyramine (3-MTA), 5-hydrooxyindoleacetic acid (5-HIAA) for their direct effect on the testosterone production by isolated interstitial cells from mice. The finding reveal that DA, 5-HT at lower doses can acutely stimulate while DOPAC, E, 3-MTA inhibits testosterone production significantly over the basal level (p<0.05). At longer duration of incubation the stimulation and inhibition of either NTs is not significantly different then the basal except for high dose of mg/ml 5-HT which showed significant inhibition both at 4 and 24 hours of incubation over the basal level. The exact mechanism how some of these putative NTs can acutely stimulate testosterone production while others can inhibit is a matter of further investigation. It is postulated that enzymatic metabolization of 5-HT to 5-HIAA and DA to 3-MTA is partly responsible for the significant inhibition of testosterone production acutely and at longer duration of incubation as seen with higher dose. Further investigation is necessary before we can come to any conclusive decision regarding their exact mechanism but NT=s local role in testosterone production from Leydig cells is an crucial clue to unfold the unique mystery of testosterone synthesis.