Although macroprolactinemia due to antiprolactin (anti-PRL) autoantibodies is not uncommon among hyperprolactinemic patients, the pathogenesis of such macroprolactinemia is still unknown. We examined IgG subclasses of anti-PRL autoantibodies by enzyme immunoassay, and PRL phosphorylation and isoforms by Western blotting, mass spectrometry, and two-dimensional electrophoresis in six patients with anti-PRL autoantibodies and in 29 controls. PRL-specific IgG subclasses in patients with anti-PRL autoantibodies were heterogeneous, but five of six patients showed IgG4 predominance, which is known to be produced by chronic antigen stimulation. Western blot and mass spectrometric analyses revealed that human pituitary PRL was phosphorylated at serine 194 and serine 163, whereas serine 163 in serum PRL was dephosphorylated. On two-dimensional electrophoresis, serum PRL mainly consisted of isoform with isoelectric point (pI) 6.58 in control hyperprolactinemic patients, whereas acidic isoforms (pIs 6.43 and 6.29) were also observed in patients with anti-PRL autoantibodies. Our data first demonstrate that human pituitary PRL is serine phosphorylated and partially dephosphorylated in serum, and suggest that the acidic isoforms may give rise to chronic antigen stimulation in patients with anti-PRL autoantibodies.