Background/Aim. Sub-Tenon's block is nowadays commonly used in ophthalmic surgery because of its safety and efficacy. The aim of this study was to investigate the distribution of the anesthetic solution with different amounts of hyaluronidase in the retrobulbar space, following an injection into the Sub-Tenon's space. Methods. In this experimental study, 40 pig cadaver heads were used (80 eyeballs). The material was divided into four groups (of 20 eyeballs each). Each group was administered 4.5 ml of a mixture of 2% lignocaine, 0.5% bupivacaine, and 0.5 ml of Indian ink, with different amounts of hyaluronidase - 15 IU/ml, 75 IU/ml, 150 IU/ml, except the control one. Samples of retrobulbar tissue were analyzed using the standard histopathological procedure. After that, they were also analyzed using the Adobe Photoshop program® (Windows, USA). The retrobulbar space was divided into eight zones by four perpendicular lines, which crossed in the centre of the optic nerve. The presence of ink in fat and muscle tissues and in the sheath of the optic nerve was observed. Results. The presence of the local anesthetic solution was significantly higher in inferonasal and superonasal quadrants of the fat and muscle tissues (p < 0.01). The distribution in optic nerve sheath is similar in each quadrant. Distribution of local anesthetic in each zone of the muscle tissue (I-VIII) was strongly influenced by the amount of hyaluronidase added. In the fat tissue, the distribution of local anesthetic under the influence of hyaluronidase was significantly higher (p < 0.05) in the areas which were distant from the place of injection (I-IV). The distribution in the optic nerve sheath is significantly higher (p < 0.01) in the group with 150 IU/ml of hyaluronidase. Conclusions. Following a sub-Tenon block local anesthetic was present in the retrobulbar space in a high percentage of the cases. The presence of local anesthetic solution in retrobulbar space depends on the amount of hyaluronidase previously added to the local anesthetic.