The significance of probe design for laser Doppler flowmetry measurement depth and variation was investigated by determining flux versus pressure relations and spatial variability with various probes placed on the serosal surface of porcine sigmoid colon. For two large probes (three optical fibers with diameter and center separation > or = 500 microns) the flux versus pressure relations showed autoregulation, indicating that the mucosal perfusion contributed to the flux signal. The curves were reproducible between individuals. With two smaller probes linear flux versus pressure relations were obtained, indicating a shallow measurement not influenced by the autoregulated mucosal perfusion. The spatial variation expressed as coefficients of variation of repeated recordings at each pressure level was significantly greater with the smaller probes (p < 0.001). For clinical use the larger probes should be preferred. These include all wall layers, and only 3 repeated recordings are needed to obtain a reproducible mean flux value within +/- 10%, as opposed to 10 with the smaller probes.