The mechanisms underlying exercise-induced increases in adipose tissue blood flow and lipolysis involve both β-adrenergic receptor (βAR)- and natriuretic peptide receptor (NPR)-dependent processes. We hypothesized that daily wheel running (RUN) would increase the expression of NPR1, NPR2, βAR2 and βAR3 in retroperitoneal (RP) and epididymal (EPI) adipose tissues of obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Four-week-old OLETF rats were assigned to sedentary (SED, n = 6), calorie-restricted (CR, n = 8; fed 70% of SED) or RUN groups (n = 8). Rats were killed at 40 weeks of age. By design, body weight and adiposity were similar between RUN and CR animals, but each was lower than SED (P < 0.01). Compared with SED, RP depots of RUN rats exhibited 1.7- to 3.2-fold greater NPR1, NPR2, βAR2 and βAR3 mRNA levels (all P < 0.05). There were no differences between CR and SED in the expression of these genes in RP adipose tissues, and there were no differences in gene expression among groups in EPI adipose tissues. At the protein level, βAR2 and βAR3 were elevated in RUN and CR groups relative to the SED group in RP adipose tissues. In order to gain insight into the mechanisms underlying the activity-induced increases in NPR and βAR mRNAs, RP adipose tissue explants from Wistar rats were treated with atrial natriuretic peptide (ANP), adrenaline and/or S-nitroso-N-acetyl-dl-penicillamine (SNAP; a nitric oxide donor) in organ culture experiments. SNAP synergistically enhanced adrenaline- and ANP-stimulated increases in NPR2 and βAR2 mRNA levels. Our data suggest that physical activity-induced increases in nitric oxide interact with adrenaline and ANP to trigger the induction of NPR and βAR mRNAs in the RP adipose tissue depot of the OLETF rat.