We investigated the chromatographic pattern of serum prolactin in 41 patients with prolactinoma and correlated the distribution of immunoreactive prolactin with the clinical variables sex, tumour size, age, and response to bromocriptine therapy. In addition, the effect of long-term storage and repeated freezing and thawing on the different molecular weight forms of prolactin was evaluated. Gel chromatography (column 100 cm X 1.5 cm) was performed in 0.1 mol/l phosphate buffer, pH 7.5, using Ultrogel ACA 54 (LKB). No correlation of age or the response to drug therapy to the elution pattern of prolactin was found. Females showed a higher percentage of big prolactin than males (10.4 +/- 1.2% vs 6.8 +/- 0.7%, mean +/- SEM, P less than 0.05) and patients with microprolactinomas too had a higher percentage of big prolactin than those with macroprolactinomas (11.3 +/- 1.8% vs 7.7 +/- 0.7%, P less than 0.05). Serum samples kept frozen for more than 2 years showed a higher percentage of bigbig prolactin (P less than 0.01) than samples stored for less than 12 months suggesting formation in vitro. However, examination of fresh samples prior to freezing also demonstrated bigbig prolactin, indicating that bigbig prolactin circulates in vivo. Repeated freezing and thawing of bigbig prolactin led to almost complete interconversion to little prolactin without any increase in immunoreactivity. This finding supports the concept that bigbig prolactin represents little prolactin loosely associated to a carrier molecule.