We examined the effect of chlorothiazide (CTZ) on the tubuloglomerular (TG) feedback system in anesthetized Sprague-Dawley rats. During infusion of CTZ (0.25 mg.kg body wt-1.min-1) we found that whole kidney glomerular filtration rate (GFR) decreased by 19% (1.0 +/- 0.1 vs. 0.8 +/- 0.1 ml/min; P less than 0.005). To asses the activity of the TG feedback system during CTZ administration we compared measurements of single-nephron (SN)GFR from tubule fluid sampled separately at proximal and distal sites. During CTZ administration, distally measured SNGFR decreased significantly by 16% (27.3 +/- 1.3 vs. 22.9 +/- 1.1 nl/min; P less than 0.025), whereas proximally measured SNGFR was unchanged. Thus the difference in SNGFR between proximal and distal determination increased during CTZ infusion (4.7 +/- 0.7 vs. 7.7 +/- 0.7 nl/min; P less than 0.025), indicating that CTZ suppresses GFR by TG feedback. Na, K, and Cl concentrations measured in the late proximal tubule fluid during control and CTZ infusions were similar. In early distal tubule fluid samples K and Cl concentrations were unaffected by CTZ infusion, whereas Na concentrations increased by 32% (47.9 +/- 2.7 vs. 63.1 +/- 2.4 mM; P less than 0.001). Proximal tubule microperfusion with 1.0 mM CTZ decreased transport rates of Na and water by approximately 40%, whereas the transport rate of Cl was not affected. In conclusion our results indicate that CTZ reduces GFR by activating TG feedback. The mechanism by which this occurs is in part due to an increase in the strength of the signal.