1. Colonic afferent fibres were recorded using a novel in vitro preparation. Fibres with endings in the colonic mucosa are described, along with those in muscle and serosa, and their responses to a range of mechanical and chemical luminal stimuli. 2. Mechanical stimuli were applied to the tissue, which included stretch, blunt probing of the mucosa and stroking of the mucosa with von Frey hairs (10-1000 mg). Chemical stimuli were applied into a ring that was placed over the mechanoreceptive field of the fibre; these were distilled water, 154 and 308 mM NaCl, 100 microM capsaicin, 50 mM HCl, and undiluted and 50% ferret bile. 3. Recordings were made from 52 fibres, 12 of which showed characteristics of having endings in the mucosa. Mucosal afferents were sensitive to a 10 mg von Frey hair and were generally chemosensitive to >= 1 chemical stimulus. 4. Ten fibres showed characteristics of having receptive fields in the muscular layer. These fibres responded readily to circumferential stretch, as well as to blunt probing. 5. Twenty-seven fibres showed characteristics of having endings in the serosal layer. They adapted rapidly to circumferential stretch and responded to blunt probing of the serosa. Fifteen of 19 serosal fibres tested also responded to luminal chemicals. 6. Three fibres were unresponsive to all mechanical stimuli but were recruited by chemical stimuli. 7. This is the first characterization of colonic afferent fibres using an in vitro method and the first documentation of afferent fibres with their endings in the mucosa of the colon. These fibres are likely to be important in aspects of colonic sensation and reflex control.