In recent studies we suggested that macula densa cytoplasmic calcium may serve in the transmission of feedback signals. This was based, in part, on the finding that the calcium ionophore A23187 in the presence of perfusate calcium enhanced the feedback responses obtained with a 70 mosmol/kg hypotonic solution. The purpose of the present studies was to further evaluate the role of calcium in the transmission of feedback signals and, in particular, to determine the source for the increase in cytosolic calcium during feedback responses. For these experiments, stop-flow pressure (SFP) feedback responses were evaluated in rats during retrograde microperfusion from an early distal tubular site at 15 nl/min. During retrograde perfusion with an isotonic Ringer solution, alterations in perfusate calcium concentration (from 4 to 0 meq/liter) or addition of 10 -3 M verapamil did not change the magnitude of the SFP feedback responses, which averaged between 12 and 13 mmHg. However, after 5 min of perfusion with the isotonic Ringer's solution containing 8-(N,N-diethyl-amino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative inhibitor of intracellular release of bound calcium, SFP feed-back responses were reduced by 57 ± 7% at 100 μM TMB-8, 80 ± 5% at 300 μM TMB-8, and 76 ± 6% at 500 μM TMB-8. The addition of 5 μM A23187 to the 500 μM TMB-8 solution restored feedback responses to control values. Accordingly, these results further support the view that calcium may be involved in the transmission of tubuloglomerular feedback signals. Furthermore, the source for the increase in cytosolic calcium during the transmission of feedback signals may be mobilization of calcium from bound or sequestered stores.