Leptin is a protein synthesized and secreted primarily by adipose tissue. The blood leptin concentration is known to reflect body fat content in rodents, humans and dogs, and thereby is useful for quantitative assessment of obesity. In the present study, we produced recombinant feline leptin in Escherichia coli transfected with feline leptin cDNA we cloned previously. The recombinant feline leptin with a molecular weight of 16 kDa induced phosphorylation of the signal transducers and activators of transcription 3 (STAT3) protein in the cells expressing rat leptin receptor. The anti-feline leptin antibody raised in rabbits reacted well to feline and human leptin and less to rodents' leptin in Western blot analysis. Sandwich enzyme-linked immunosorbent assay (ELISA) was developed, using rabbit anti-feline leptin antibody and recombinant feline leptin as a standard. In this ELISA system, cross-reactivity to human, rat and mouse leptin was 30.7%, 69.5% and 66.6%, respectively. The plasma leptin levels of 24 healthy cats were in a range from 0.3 to 29.7 ng/ml with the mean +/- SEM of 4.5 +/- 1.3 ng/ml, being positively proportional to body fat content. These results indicate that our ELISA system may be useful for assessment of obesity in cats.