The study was undertaken to analyze the lipolytic effect and transcriptional regulation of tumour necrosis factor (TNF)alpha in gilthead sea bream (Sparus aurata L.). The study was also focused on the transcriptional regulation and analysis of the 5-flanking region of lipoprotein lipase (LPL) in an attempt to identify cis-regulatory elements that would support the TNFalpha-mediated effects. The lipolytic effect of TNFalpha was evidenced by the increased release of glycerol in the culture medium of freshly isolated adipocytes. This observation, in addition to the summer up-regulation of TNFalpha transcripts in liver and mesenteric adipose tissue, supported a key role of TNFalpha as a fish limiting factor of tissue fat mass. Accordingly, TNFalpha expression in liver and mesenteric adipose tissue was reduced by fasting. Furthermore, the up-regulated expression of TNFalpha in the skeletal muscle of older fish can represent an adaptive response to limit the enhanced lipid influx towards muscle. A close positive association between LPL and TNFalpha transcripts supported the contribution of TNFalpha as a part of a regulatory network that exerts an inhibitory tonus upon the expression of LPL, which in turns limits the tissue uptake of fatty acids and the ultimate increase of tissue lipid reservoirs. The precise mechanism for the inhibition of LPL gene expression by TNFalpha remains to be established in fish, but analysis of the 5'-flanking region evidenced the conservation through vertebrate evolution of a functional OCT-1/NF-Y site that would mediate the TNFalpha effects on LPL expression.