The aim of the study was to evaluate the presence of aromatase cytochrome P450 enzyme (P450AROM) expression in normal pituitary tissues and tumor tissues of patients with prolactinoma and to examine the impact of the P450AROM expression on clinical outcome. Twenty-six consecutive human pituitary tissue samples were obtained from autopsies performed at the Institute of Forensic Medicine. Sixty-four patients who had an adenomectomy between 2000 and 2009 after prolactinoma diagnosis with histologically confirmed pituitary tumor tissues were retrospectively included in this study. The slices from the pituitary tissues were subjected to immunohistochemical staining for evaluation of P450AROM and estrogen receptor beta (ER beta) subunit. Immunohistochemistry results were compared according to age, gender, remission rate, resistance and invasion status of the patients. Higher than normal P450AROM expression was found in the pituitary tissues of the patients with prolactinoma (p < 0.001). P450AROM intensity had no relation to resistance or remission in patients with prolactinoma (p = 0.44, p = 0.45, respectively). The subgroup analysis showed that compared to males without invasive adenoma, males with invasive adenoma had higher P450AROM expression (p = 0.048). ER beta was found to have an impact on resistance (p = 0.049). This study shows that P450AROM expression is present in the pituitary tissues of patients with prolactinoma and that this presence could be important in development and tumor behavior of prolactinomas.