Previously, we have suggested that a cytosolic calcium system participates in the transmission of signals between distal tubular fluid and the glomerular vascular elements. Since the Ca-binding protein calmodulin has been implicated in the intracellular actions of cytosolic Ca, we evaluated the effects of the calmodulin antagonists trifluoperazine (TFP) and calmidazolium (R24571) on tubuloglomerular feedback responses. In the rat, stop-flow pressure (SFP) was measured during retrograde perfusion into the distal tubule at 15 nl/min for 5 min. Perfusion with an isotonic Ringer solution resulted in a decrease in SFP of 12 ± 1.1 mmHg (n = 34). With the addition of 50-75 μM TFP, SFP was decreased by 11 ± 1.0 mmHg (n = 24); with 500 μM TFP, SFP feedback responses were 10 ± 1.2 mmHg (n = 7). During perfusion with 20 μM R24571, SFP decreased by an average of 12 ± 1.1 mmHg (n = 18). Single-nephron glomerular filtration rate feedback responses during retrograde microperfusion were also unaltered by R24571 (Δ15 ± 2.5 vs. Δ14 ± 2.6 nl/min). In the SFP experiments it was observed that the recovery of SFP toward preinfusion values after cessation of perfusion was significantly prolonged in tubules perfused with the calmodulin antagonists. SFP recovery times averaged 65 ± 5.5 s (control), 104 ± 9.9 s (50-75 μM TFP), 154 ± 9.6 s (500 μM TFP), and 116 ± 9.9 s (20 μM R24571). Accordingly, these results suggest that activation of calmodulin is not required for the transmission of tubuloglomerular feedback signals. Since calmodulin can stimulate Ca transport, the delay in the return of the SFP to control values in tubules perfused with calmodulin antagonists may be due to an impaired ability of the macula densa cells to rapidly lower cytosolic Ca.