Background: Methamphetamine (MA) use disorders are pervasive global social problems that produce large medical and public health burdens. Abnormalities in pituitary hormonal regulation have been observed in preclinical models of substance abuse and in human substance abusers. They have, however, not been studied before in MA-dependent human subjects. Objectives: To determine if MA-dependent research volunteers differ from healthy control subjects in plasma levels of adrenocorticotropic hormone (ACTH), cortisol, or prolactin, or in pituitary dopamine D2 receptor availability during early abstinence from MA. Methods: MA-dependent subjects (N = 31), who were not seeking treatment, resided on an inpatient ward for up to 5 weeks. Abstinence was confirmed by daily urine drug screening. Venous blood was sampled for plasma hormone levels, and positron emission tomography with [18F]fallypride was performed to determine dopamine D2 receptor availability during the first week of abstinence. Venous blood was sampled again for hormone levels during the fourth week of abstinence. Matched healthy volunteers (N = 23) participated as a comparison group. Results: MA-dependent and healthy comparison subjects did not differ in plasma ACTH or cortisol levels, but had an elevated plasma prolactin at both the first week and fourth week of abstinence. There was no group difference in pituitary dopamine D2 receptor availability. Conclusion: MA-dependent individuals have abnormalities in prolactin regulation, which is not likely due to alterations in pituitary dopamine D2 receptor availability. Scientific significance: MA dependence is associated with elevated prolactin levels, which may contribute to medical comorbidity in afflicted individuals.