Although a wealth of evidence exists indicating that proteolytic cleavage can enhance the biological activity of the growth hormone (GH) molecule, the mechanisms responsible for the generation of GH fragments are not completely understood. In the present work we investigated the ability of different rat tissues to cleave 22 kDa GH, as well as the influence of sex and age, the two major physiological regulators of GH secretion on this process. Our results show that tissue homogenates obtained from rat liver, skeletal muscle or adipose tissue (three well-documented target organs for the hormone) are able to cleave 22K-GH, while the hormone is resistant to cleavage by rat brain homogenates. This process is rather selective for 22K-GH, since the 20 kDa GH variant exhibits stability to degradation by all tissue homogenates investigated. Moreover, only a minor fraction of 22 kDa GH is cleaved under our experimental conditions, suggesting that GH microheterogeneity within the 22 kDa range may also determine hormone susceptibility. Finally, we also found that 22K-GH processing shows important age-related changes (the greatest intensity observed in 4-day-old pups), while no gender-related differences exist in any of the tissues investigated.