A 41-year-old man was diagnosed with a prolactinoma on account of a significantly elevated serum prolactin and a large pituitary lesion. He was managed with dopamine agonist therapy. Although serum prolactin had normalised, tumour shrinkage was not achieved, and he subsequently underwent transsphenoidal resection of the mass. An unexpected finding of negative prolactin immunostaining was noted on pathological evaluation of the tumour specimen. Postoperative serum prolactin remained in the normal range. Three years later, we performed further immunohistochemical analysis of the adenoma. The lack of prolactin expression was reconfirmed, but positive staining for the transcription factor Pit-1 was observed, supporting the initial diagnosis of a lactotroph adenoma. We draw attention to the fact that absence of tumour prolactin content in prolactin secreting macroadenomas is uncommon. If serum prolactin is only mildly or moderately elevated, distinguishing a prolactinoma from a non-functioning pituitary macroadenoma associated with stalk compression can be difficult. Determination of Pit-1 expression is therefore a crucial step in establishing a diagnosis in this scenario.