The morphology and function of Leydig cells are changed during the development, mature and senility of Leydig cells along the life span of males. This study was to observe the growth morphology of adult mouse Leydig cells in culture, aiming to provide a reference for furthermore understanding of the biological function of Leydig cells by in vitro model. Testes of two-month-old mice were decapsulated and then the Leydig cells were isolated by collagenase digestion and were cultured in DMEM/F12 supplemented with 10% FBS. The Leydig cells were identified by HSD3B staining and RT-PCR. After 48-h Leydig cell culture, both the nucleus and the cytoplasm were very clear under the optical microscope. The nucleus was big and round and the cytoplasm was filled with abundant lipid drops with a strong refractivity. After 5-day culture, Leydig cells were fully elongated in spindle, triangular, polygonal, oval or irregular shapes. Some cells grew in aggregation, and some cells grew independently. Leydig cells in aggregation elongated many cellular tentacles for intercellular connections, which formed an epithelium-like appearance. After HSD3B staining, the individual Leydig cells were stained with different extents, demonstrated that the heterogeneity of HSD3B activity in individual Leydig cells in primary culture. RT-PCR results showed that Leydig cells in culture after 5 days could express Leydig cell-specific transcriptions, HSD3B6, CYP17A1 and StAR. These results showed the morphological characterization of adult mouse Leydig cells in culture, which will lay a foundation to elucidate the relationship between the morphology and function of Leydig cells. (C) 2015 Elsevier Inc. All rights reserved.