Cholecystokinin (CCK) is a hormone secreted by the proximal small intestine and mediates pancreatic exocrine secretions in response to luminal nutrient flows. Glutamic acid and other amino acids can increase secretions of CCK among mice, dogs, and STC-1 cells. Further, inosine 5′-monophosphate (IMP) can increase amino acid induced CCK release from murine proximal intestine. Our objective was to quantify effects of IMP on glutamic acid induced CCK release from bovine small intestine. Small intestines were collected at slaughter from 2 steers (638 ± 3.2 kg BW) fed a common dry rolled corn-based diet. The small intestine was measured after removal of digesta, and 1-m sections of duodenum, jejunum and ileum were collected. Each small intestinal section was cut longitudinally, rinsed and serosa was removed before collection of mucosa. Mucosal explants (1-cm in diameter) were incubated (37°C) in HBSS (containing 1.26 mM Ca²⁺) with 20 mM HEPES (pH 7.4) and supplemented with 0, 10, 20, or 30 mM glutamic acid and 0 or 2.5 mM IMP. After 1 h, incubation buffer was collected, centrifuged to remove cellular debris, and frozen (−80°C) before analyses. Cholecystokinin secretions were quantified by radioimmunoassay. Cholecystokinin release was greater (P < 0.01) from duodenum (68.5 ± 5.0 pM) than from jejunum (8.5 ± 4.9 pM) and ileum (2.3 ± 4.9 pM). Release of CCK was 529% greater (glutamic acid × IMP <0.01) when proximal small intestine was supplemented with glutamic acid and IMP (2.79 pM CCK released / mM glutamic acid) than when proximal small intestine was supplemented with glutamic acid alone (0.53 pM CCK released / mM glutamic acid). Cursory comparisons to previous data suggest that glutamic acid induced CCK release by proximal intestine is less in cattle than in mice; however, IMP enhanced glutamic acid induced CCK release in cattle to a greater extent than previously reported in mice.