Summary:The fractional vitamin B12 absorption (FAB12) was determined in 39 patients with various gastrointestinal diseases by a double‐isotope technique, employing a single stool sample test (SSST), as well as a complete stool collection. The age of the patients ranged from 2.5 months to 16.2 years (mean 5.0 years). The test dose was administered orally and consisted of 0.5‐4.5 μg of 57CoB12 (approximately 0.05 μCi), carmine powder, and 2 mg 51CrCl3 (approximately 1.25 μCi) as the inabsorbable tracer. The whole‐body radiation to a 1‐year‐old child averaged only 20 mrad. The stool and napkin was collected and homogenized by addition of 300 ml “chromium sulfuric acid.” A 300‐ml sample of the homogenized stool and napkin, as well as 300 ml chromium sulfuric acid (75% v/v) containing the standards, were counted in a broad‐based well counter. The FAB12 determined by SSST employing the stool with the highest content of 51Cr (which corresponded to the most carmine‐colored stool) correlated closely to the FAB12 based on complete stool collection (r = 0.98, n = 39, p < 0.001). The reproducibility of FAB12 determined by SSST was assessed from double assays in 19 patients. For a mean value of 12%, the SD was 3%, which corresponded to a coefficient of variation (CV) of 25%. The excretion of 57Co and 51Cr in the urine was examined in six patients with moderate to severe mucosal damage and was found to be low. The gut transit time, as determined by the excretion time of ≥ 50% of the orally administered 51Cr, ranged from 4 to 100 h (mean 36 h, n = 39), and it was less than 12 h and 24 h in 7 and 13 patients, respectively. Conclusively, the SSST proved to be a very suitable technique for measurements of B12 absorption, even in children with mucosal damage of the intestine and short gut transit time. Further‐more, the method fulfilled the following requirements; (a) minimal cooperation and control of possible errors, (b) small radiation dose, (c) direct measurement of the B12 absorption, and (d) no need for flushing with B12.