Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism of the mammalian kidney (1). mUT-A1 and mUT-A3 are facilitative urea transporters that are expressed in mouse inner medullary collecting duct cells (2). The mIMCD-3 cell line is an osmotically-tolerant mouse cell line derived from the terminal part of the mouse inner medullary collecting duct (3). Using a previously characterised antibody (ML446) (2), immunoblotting experiments detected ~45, ~60 and ~120 kDa mUT-A3 signals in protein samples from mIMCD-3 cells grown under ‘normal’ osmotic conditions (~300mOsm). Growing cells for up to two weeks in hyperosmotic media containing additional NaCl and urea (either ~500, ~700 or ~900mOsm) significantly reduced mIMCD-3 growth rates (P<0.05, ANOVA, n = 3). However, there was a significant increase in expression levels of mUT-A3 (P<0.05, ANOVA, n = 3). In contrast, no signals were detected in any samples using a mUT-A1-specific antibody (ML194) (2), showing that mIMCD-3 cells do not express mUT-A1. This study shows that mIMCD-3 cells express mUT-A3 but not mUT-A1 urea transporters. The mIMCD-3 cell line is therefore a suitable model for studying the effects of hyperosmolality on expression of mUT-A3 urea transporters.