Testes and ovaries of Enteroctopus megalocyathus collected along the Patagonian Atlantic coast were analysed histologically to validate the macroscopic maturity scales adopted for this species. Changes through the course of development of the seminiferous tubules and of the oocyte/follicular cell complexes were characterized and these were classified into five and six microscopic categories of development respectively. A histological maturity index, based on the frequencies of microscopic categories, was used to assess the correspondence between macroscopic maturation stages and the microscopic level of development of the gonadal tissue. Seminiferous tubules showed a regular and progressive pattern of microscopic development within each macroscopic stage and between consecutive macroscopic stages. However, a minority of males exhibiting seminiferous tubule with sperm did not display macroscopic characteristics of the mature-spawning stage. In females, an overlapping of microscopic categories was observed in maturing macroscopic stages. Previtellogenic oocytes were not present at mature-spawning or spent stages. Significant changes in the histological maturity index were observed between consecutive macroscopic stages, confirming the validity of macroscopic maturity scales of both sexes. In addition, by considering both macroscopic and microscopic criteria, it was possible to determine the overall state of development and functioning of the reproductive system during sexual maturation of this species.