Ultrasound imaging was applied, for the first time, in the examination of the central nervous system of the cephalopod mollusc Octopus vulgaris, an invertebrate. Goals of this study were: i. to reveal and measure the cerebral masses in vivo, in their anatomical position; ii. to evaluate and compare the dimensions of the different parts of the octopus brain in vivo and postmortem, and iii. to test the reproducibility of the ultrasound method both in reaching a given sonographic plane in the same individual at two different times and in evaluating potential changes in brain size due to animal growth. Our results show that ultrasonography is a reliable method to measure the various parts of the octopus brain. Sonographic measurements of the brain masses in vivo were correlated with those determined postmortem. In addition, brain size estimation is reproducible via ultrasound: no significant difference resulted when measurements of the same brain were taken over consecutive days. Furthermore, when the time lapse between the two sonographic examinations was long enough (30 days), we were able to detect changes in brain dimensions in the same octopus.