The present study using secretagogue substances was undertaken to investigate Harderian gland secretion in the frog, Rana esculenta. Carbamylcholine chloride and bethanechol injections caused enhancement of the secretory activity and hyperemia, while nicotine did not. Morphological examinations showed reduced cellular height and dilated alveolar lumina, containing secretory granule discharge, nuclei and cytoplasmic fragments, indicating an apocrine and holocrine secretion type. The administration of atropine prevented the enhancement of the secretion. Our data suggest that cholinergic stimulation provokes enhancement of the secretory activity on the frog HG, and this mechanism appears to be mediated by the activation of the muscarinic receptors.