Adipocytes were recently identified as an important source of endocrine and paracrine mediators, regulating the metabolism and activity of various cell types and body functions. 3T3-L1 preadipocytes are useful model for physiological, pharmacological and cell signaling studies. Differentiation of 3T3-L1 murine fibroblasts into adipocyte-like cells was conducted in presence of IBMX, dexamethasone and insulin and demonstrated by Oil Red O staining of accumulated lipid droplets. Using Inflammatory Multi- Analyte Cytokines ELISArray Kit we investigated the release of cytokines under basal conditions, after PGF2α treatment for 24 hours to induce pro-inflammatory phenotype, and after PGF2α treatment and incubation in the presence of L-C-Propargylglycine (PGG, 1 mmol/l), a selective inhibitor of cystathionine-gamma-lyase (CSE). The last combination was used to explore the role of H2S, released from CSE, for cytokine and H2O2 release. We found that PGF2α strongly increased TNFα secretion from differentiated adipocytes, the latter effect being antagonized by PGG. The CSE inhibitor enhanced IL-6 production and suppressed IL-10 secretion. PGG enhanced H2O2 production of in PGF2α-treated cells. It is concluded that pro-inflammatory phenotype of differentiated 3T3-L1 adipocyte-like cells, induced by PGF2α is characterized by enhanced TNFα production which critically depends on the ability of CSE to produce H2S.