SUMMARY: A procedure for estimating rat prolactin and growth hormone (somatotrophin, STH), by measuring the optical density of the electrophoretically isolated and stained hormone bands in polyacrylamide gel columns, is described and evaluated. A simple and inexpensive densitometer is also described. Prolactin levels in adenohypophyses and in medium from pituitary incubates were measured by electrophoresis-densitometry (ED) and by the pigeon crop-sac assay. The two methods showed a high degree of correlation. The validity of the ED method for estimating prolactin levels in adenohypophysial tissue and in incubation medium was demonstrated by comparing the prolactin content of adult male and female and of oestrogen-treated male glands and by experiments in vitro. The female pituitary contained about three times more prolactin than the male and the glands of oestrogen-treated males had levels about the same as those of females. It was also shown that the ED method could be used to demonstrate the inhibitory effects of extract of rat hypothalamic tissue on prolactin secretion in vitro by the rat pituitary. Levels of STH in adult male glands, as measured by this method, were comparable to results obtained by others using immunoassays. Propylthiouracil-induced hypothyroidism depressed the STH and prolactin levels in male rat pituitaries, in agreement with the observations of others. The stainability of the prolactin band in rat adenohypophyses was observed to decrease with time when the glands were stored on dry ice. No such change occurred in the staining characteristics of the STH band. Other aspects of the ED method are discussed, including its precision, efficiency, sensitivity, economy and utility.