We injected acetazolamide (AZ; 5 x 10(-6) M; 1 nl; n = 14), its inactive analogue 2-acetylamino-1,3,4-thiadiazole-5-sulfon-t-butylamide (5 x 10(-5) M; n = 6), or mock cerebrospinal fluid (n = 5) into the caudal raphe in the midline brain stem of anesthetized paralyzed ventilated rats. These AZ injections have been shown to produce a focal region of tissue acidosis with a radius < 350 microns and are used as a probe for sites of central chemosensitivity. Compared with control injections, AZ injection into the raphe, as demonstrated by anatomic analysis of injection location, significantly increased the amplitude of the integrated phrenic neurogram over 10-40 min. Not all raphe injections produced such a response. AZ injections identified as responders (n = 8 of 14) increased integrated phrenic amplitude 43.3 +/- 10.7% (SE) of baseline 20 min after the injection. We conclude that the midline caudal raphe contains sites of ventilatory chemoreception.