A new clonal strain of Prl-secreting cells derived from the transplantable rat pituitary tumour, 7315a, has been established in culture. The cells of this strain, designed 235-1, have a highly developed Golgi complex, an extensive rough endoplasmic reticulum, and a few small but no large dense-core granules. When inoculated into athymic mice and rats of the Buffalo strain, the 235-1 cells produce tumours, and the host animals have hypertrophied mammary glands that produce milk, indicating that Prl secreted by these cells has mammotrophic activity. In monolayer culture, the doubling time of 235-1 cells is 31 +/- 1 h (mean +/- SE). The cells secrete Prl, a trace quantity of GH, but no LH, FSH, TSH, ACTH, or alpha-MSH. Prl is released at a rate of 257 +/- 12 fg per h per cell. The cellular content of Prl is 424 +/- 23 fg per cell. Prl secretion by 235-1 cells is not affected by dopaminergic agonists and antagonists, TRH, or oestradiol-17 beta but is inhibited in the presence of EGTA or monensin, an ionophore that is believed to act at the level of the Golgi complex. The subcellular distribution of Prl in 235-1 cells is different from that in rat pituitary cells. In 235-1 cells, Prl is associated not with a single set of dense particles as it is in pituitary cells but with 2 sets of subcellular particles, of which 1 set cosedimented with particles having lysosomal enzyme activity. These findings suggest that Prl secretion by 235-1 cells involves secretory pathways that are different from those seen in normal lactotrophs.