Abnormalities in transepithelial electrolyte transport in cystic fibrosis rectum were analyzed by short-circuit current measurements on 11 control subjects and 11 subjects with cystic fibrosis in a modified Ussing chamber. As judged by the amiloride-sensitive component of the short-circuit current, electrogenic sodium absorption appeared unmodified in cystic fibrosis. In contrast, the shortcircuit current response to specific stimuli of both cyclic adenosine monophosphate (cAMP)- and calcium-mediated chloride secretion was drastically altered in all of the cystic fibrosis biopsy specimens examined. Stimulation of the cAMP pathway by 8-bromo cAMP or forskolin resulted in a sustained increase in short-circuit current in control tissues (+2.51 ± 0.63 μA/cm²) but in a slight change in the opposite direction in cystic fibrosis (−0.56 ± 0.49 μA/cm²; P < 0.05). Carbachol, a calcium-linked secretagogue, provoked a transient increase in short-circuit current in all of the control tissues (peak response, +26.69 ± 3.63 μA/cm²) but a transient change in the opposite direction in 6 of 11 cystic fibrosis tissues (−12.46 ± 4.64 μA/cm²; P < 0.05). In 2 of 11 patients with cystic fibrosis, however, a significant but subnormal and transient increase in short-circuit current was observed (+2.62 ± 0.04 μA/cm²; P < 0.05), whereas in 3 of 11 patients with cystic fibrosis a transient change in the opposite direction (−9.83 ± 2.20 μA/cm²; P < 0.05) was followed by a small and transient increase (+2.89 ± 0.83 μA/cm²; P < 0.05). Using the calcium-mediated secretory response therefore, patients with cystic fibrosis could be divided into two categories: a major population showing defective anion secretion but active cation secretion and a subclass (including three siblings) showing residual but subnormal anion secretion. The easy accessibility of rectal samples and the inversed direction of the cAMP- or calcium-provoked short-circuit current is of considerable advantage in the diagnosis of cystic fibrosis.