Abstract The occurrence of atrial natriuretic factor (ANF) immunoreactivity was investigated in the adrenal gland of the lizard Podarcis sicula by avidin-biotinylated peroxidase complex (ABC) immunocytochemical technique: ANF immunoreactivity was present in the chromaffin tissue, and was absent in the steroidogenic tissue. The role of ANF in the modulation of the pituitary–adrenal axis activity was investigated in vivo by intraperitoneal administration of ANF. The effects were evaluated by examination of the morphological and morphometrical features of the tissues, as well as the plasma levels of adrenocorticotropic hormone (ACTH), corticosterone, aldosterone, norepinephrine, and epinephrine. ANF (28 μg/100 g body wt) did not affect ACTH plasma levels, that remained almost unchanged; in contrast, corticosterone plasma levels increased from 6.45 ± 0.070 ng/ml in carrier-injected lizards to 9.69 ± 0.080 ng/ml 24 h after the injection; aldosterone levels decreased from 2.19 ± 0.010 ng/ml in carrier-injected specimens to 0.58 ± 0.003 ng/ml 24 h after the experimental treatment. In the chromaffin tissue, an increase in the number of epinephrine cells and a decrease in the number of norepinephrine cells were observed, decreasing the numeric norepinephrine/epinephrine cell ratio, from 1.4/1 of control specimens to 0.3/1 24 h after ANF administration. Moreover, norepinephrine plasma levels decreased from 998 ± 4.600 pg/ml in carrier-injected specimens to 321 ± 2.230 pg/ml 24 h after ANF administration; epinephrine plasma levels were elevated from 614 ± 3.410 pg/ml in carrier-injected specimens to 1672 ± 10.800 pg/ml 24 h after the experimental treatment. The presence of ANF in the adrenal gland suggests that, also in reptiles as in other vertebrates, this peptide, locally released from the chromaffin cells, may modulate the activity of the adrenal gland, probably in a paracrine manner. The effects of ANF on the adrenal gland suggest that this peptide may affect reptilian salt and fluid homeostasis.