Calcium mobilization from intracellular stores represents an important cell signalling process that is regulated, in mammalian cells, by inositol-1,4,5-triphosphate (IP3), cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Intracellular calcium is important for mobilization of secretory granules to the plasma membrane in preparation for exocytosis. NAADP mobilizes calcium from lysosome-related acidic compartments and it has recently been shown that two-pore channels (TPCs) comprise a family of NAADP receptors, with TPC1 expressed on endosomal membranes. Western blot analysis and RT-PCR have revealed TPC1 expression in anterior pituitary and double-labelling immunofluorescence labelling for prolactin PRL and TPC1 demonstrated colocalisation in lactotrophs. Here we test the hypothesis that PRL secretion in lactotrophs from TPC1 null mice would be impaired and excess storage of secretory granules would result. Anterior pituitary sections from male and female wild-type (WT) and TPC1 null mice (n=4 of each) were immunogold labelled for PRL and examined by quantitative electron microscopy to determine lactotroph size, secretory granule characteristics and distribution. In male TPC1 null mice there was a significant (P<0.01 versus WT) decrease in cell and cytoplasmic area, and a significant decrease (P<0.01) in granule density suggesting decreased synthesis and storage of PRL. Furthermore, there was an increase in the percentage of secretory granules within a 300 nm margin of the plasma membrane indicating that more granules were distributed adjacent to sites of secretion (P<0.01). In female TPC1 null mice there was no significant difference measured in cell and cytoplasmic area, granule distribution or size but a significant (P<0.05 versus WT) decrease in granule density. RIA demonstrated that pituitary PRL content was significantly decreased (P<0.05 versus WT) in male and female TPC1 null mice. In conclusion, the data are consistent with dysregulation of PRL control in the absence of TPC1 but leading to decreased PRL storage in lactotrophs.