To determine if calcium‐dependent secretagogues directly act on epithelial cells to elicit CI⁻ secretion, their effects on CI⁻ transport and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl⁻ sensitive fluorescent probe, 6‐methoxyquinolyl acetoethyl ester, MQAE and the Ca²⁺‐sensitive fluorescent probe, fura‐2AM were used to assess Cl⁻ transport and [Ca²⁺]_i, respectively. Basal Cl⁻ transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl⁻ channel blocker diphenylamine‐2‐carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na⁺/K⁺/2Cl⁻ cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I⁻ > F⁻ ≥ Cl⁻ > Br⁻. DPC inhibited I⁻ influx by ∼50%, F⁻ influx by 80%, Cl⁻ influx by 85%, and Br⁻ influx by 90%. Furosemide significantly inhibited influx of Br⁻ (84%) and Cl⁻ (81%) but not of F⁻ and I⁻. The effects of agents known to alter biological response by increasing [Ca²⁺]_i in other epithelial systems were used to stimulate Cl⁻ transport. Cl⁻ influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca²⁺ ionophore A23187 (2.05 ± 0.40). The Cl⁻ permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine‐induced Cl⁻ influx was significantly inhibited by only furosemide. Indomethacin blocked histamine‐stimulated Cl⁻ permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl⁻ transport by activating the Na⁺/K⁺/2Cl⁻ cotransporter via a cyclooxygenase‐dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl⁻ channels and the cotransporter, and their actions are cyclooxygenase‐independent. © 1996 Wiley‐Liss, Inc.