We investigated the co-localization in secretory granules of secretogranins/chromogranins, thyrotropin, and luteinizing hormone in ultra-thin frozen sections of cow anterior pituitary by double immunoelectron microscopy, using specific antibodies and protein A-gold particles of different sizes. The distribution of secretogranin II, chromogranin A, and chromogranin B (secretogranin I) was largely similar. In cells containing secretory granules of relatively small size (100-300 nm) and low electron density (identified as thyrotrophs and gonadotrophs by immunolabeling for the respective hormone) and in cells containing both small (170-250 nm) and large (300-500 nm) secretory granules of low electron density (also identified as gonadotrophs), all three secretogranins/chromogranins were detected in most if not all granules, being co-localized with the hormone. In cells containing both relatively large (400-550 nm), electron-dense granules and small, less electron-dense secretory granules (150-300 nm), identified as somatomammotrophs by double immunolabeling for growth hormone and prolactin, all three secretogranins/chromogranins were predominantly detected in the subpopulation of small, less electron-dense granules containing neither growth hormone nor prolactin. Interestingly, this granule subpopulation of somatomammotrophs was also immunoreactive for thyrotropin and luteinizing hormone. These data show that somatomammotrophs of cow anterior pituitary are highly multihormonal, in that the same cell can produce and store in secretory granules up to four different hormones and, in addition, the three secretogranins/chromogranins. Moreover, selective localization of the secretogranins/chromogranins together with thyrotropin and luteinizing hormone in a subpopulation of secretory granules of somatomammotrophs indicates the preferential co-packaging of the secretogranins/chromogranins and these hormones during secretory granule formation.