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Background: The mechanism of short-chain fatty acid (SCFA) absorption by the colon is not known. The aim of these experiments was to identify the transport mechanisms present in the basolateral membrane to develop an overall model of colonic SCFA absorption. Methods: These studies determined the uptake of [¹⁴C]-butyrate (used as a model SCFA) by basolateral membrane vesicles prepared from rat distal colonic mucosa. Results: Significantly higher [¹⁴C]butyrate uptake under an acidic environment (extravesicular pH [pH₀] = intravesicular pH [pH₁] = 5.5) than that under alkaline environment (pH₀ = pH₁ = 7.5) indicates the presence of nonionic diffusion. In the absence of a pH gradient (pH₀/pH₁ = 7.5/7.5), outward gradients of bicarbonate significantly stimulated [¹⁴C]butyrate uptake. Additional presence of a pH gradient (pH₀/pH₁ = 6.0/7.5) further enhanced the bicarbonate gradient-stimulated [¹⁴C …