In this study, the intracellular signaling mechanisms through which TNFα increases LDH(A4) activity/expression in primary cultures of porcine testicular Sertoli cells were investigated. Studies were focused on sphingomyelin hydrolysis pathway. Treatment of [¹⁴C]serine-labeled cells with TNFα (15 ng/ml, 0.8 nM) resulted in a transient decrease (∼20%) in cellular [¹⁴C]sphingomyelin and in an increase (∼27%) in [¹⁴C]sphingosine that remained elevated for at least 75 min. In the same experiments, no significant changes were detected in ceramide levels. Exogenous sphingosine stimulated LDH(A4) activity and LDHA expression in a dose-dependent manner (ED₅₀ = 8 μM of sphingosine). Such an increase in LDHA messenger RNA levels and LDH(A4) activity was detected at 24 h and was maximal after 48 h of treatment. Kinetically, the increase in LDH(A4) activity was similar whether Sertoli cells were treated with sphingosine (12 μM) or with TNFα (20 ng/ml). Although sphingosine mimicked the action of TNFα on Sertoli cells LDH(A4) activity and expression, the maximal stimulatory effect represented about 30% of TNFα maximal activity. Sphingomyelinase, C2 ceramide, sphingosine 1-phosphate, N,N-dimethylsphingosine, and phosphorylcholine had no significant effect on LDHA expression/LDH(A4) activity. Exogenous C2 ceramide increased LDH(A4) activity only in cytokine-treated cells, suggesting its involvement as sphingosine precursor in TNFα-stimulated LDH(A4) activity via the sphingomyelin hydrolysis pathway. The LDH(A4) activity stimulated by TNFα was decreased by 36.2% by an inhibitor of sphingosine formation, NH4Cl (4 mM), supporting a role of sphingosine in the TNFα effect. Moreover, bisindolylmaleimide (100 nM), a protein kinase C (PKC) inhibitor decreased significantly by 28.7% the TNFα effect on LDH(A4) activity but had no effect on the stimulating action of sphingosine, suggesting that if PKC is involved in TNFα action, the sphingosine effect on LDH(A4) is unrelated to the PKC activity or inhibition. Together, the present data suggest that in primary Sertoli cell cultures, TNFα stimulating action on LDHA expression is partly exerted via sphingomyelin hydrolysis pathway, sphingosine being the active metabolite.