Treatment for 40 h of reaggregate pituitary cell cultures from 14‐day‐old female rats with nanomolar concentrations of γ3‐melanocyte‐stimulating hormone (MSH) increased prolactin mRNA but not growth hormone (GH) mRNA expression levels as measured by quantitative real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR). During the 40 h incubation, γ3‐MSH stimulated prolactin accumulation in the culture medium. α‐MSH, a potent agonist of the rat melanocortin‐3 receptor (MC3R) and Ala⁸‐γ2‐MSH, a very weak agonist of the MC3R, increased prolactin mRNA expression at a similar concentration range as γ3‐MSH. The effect of γ3‐MSH on prolactin mRNA expression was abolished when aggregates were cultured in the presence of thyroid or glucocorticoid hormones, but not of oestradiol. By contrast, oestradiol abolished the stimulatory effect of Ala⁸‐γ2‐MSH on prolactin mRNA expression. In GH3 cells stably transfected with the enhanced green fluorescent protein (eGFP) gene under control of a 3‐kb prolactin promoter fragment, a dose as low as 1 nMγ3‐MSH, added for 24 h, significantly increased eGFP fluorescence. Agouti‐related protein (AgRP⁸³⁻¹³²), a known endogenous MC3R and MC4R antagonist, did not reduce the stimulation of prolactin mRNA expression by γ3‐MSH or Ala⁸‐γ2‐MSH. On its own, AgRP⁸³⁻¹³² significantly increased prolactin mRNA expression level and prolactin accumulation. Both γ2‐MSH and Ala⁸‐γ2‐MSH increased [S³⁵]GTPγS binding in membrane preparations of 14‐day‐old rat pituitaries and of GH3 cells. Whereas MC3R and MC5R mRNA were detectable by RT‐PCR in normal pituitary, these receptor mRNAs were undetectable in GH3 cells using various oligonucleotide primer sets. The present findings indicate that melanocortin peptides stimulate prolactin gene expression and production and that, at least in part, a receptor different from the classic MCR is involved. AgRP appears to have other actions than its known antagonistic activity on the MC3R and MC4R.