Previously we characterized localization of Na⁺-glucose cotransporter SGLT1 (<I>Slc5a1</I>) in the rat kidney using a polyclonal antibody against the synthetic C-terminal peptide of the rat protein (Sabolic et al., Am J Physiol Renal Physiol 290:913-926, 2006). However, the antibody gave some false-positive reactions in immunochemical studies. Using a shortened peptide for immunization, here we generated an improved, more specific anti-rat SGLT1 antibody (rSGLT1-ab), which in immunochemical studies with isolated membranes and tissue cryosections from male (M) and female (F) rats exhibited: a) in kidneys and small intestine, labeling of a major protein band of ~75 kDa, b) in kidneys of adult animals, localization of rSGLT1 to the proximal tubule (PT) brush-border membrane (S1<S2<S3) and intracellular organelles (S1>S2>S3), with zonal (cortex<outer stripe) and gender differences (M<F) in the protein expression, which correlated well with the tissue expression of its mRNA in RT-PCR studies, c) in kidneys of castrated adult M, upregulation of the protein expression, d) in kidneys of prepubertal rats, weak and gender-independent labeling of the 75 kDa protein band and immunostaining intensity, e) in small intestine, gender-independent regional differences in protein abundance (jejunum>duodenum=ileum), and f) thus far unrecognized localization of the transporter in cortical thick ascending limbs of Henle and macula densa in kidney, bile ducts in liver, enteroendocrine cells and myenteric plexus in the small intestine, and initial ducts in the submandibular gland. Our improved rSGLT1-ab may be used to identify novel sites of SGLT1 localization and thus unravel additional physiological functions of this transporter in rat organs.