The aim of this study was to examine the role of calcium in the release of acetylcholine (ACh) from the fetal circulation of the dually perfused human placenta. The viability of this preparation was demonstrated; Glucose consumption and lactate production (as sums of both the fetal and maternal values) over the 4 h perfusion period were 13.9 +/- 4.2 mmol/kg/h (mean +/- SEM, n = 7) and 15.1 +/- 2.0 mmol/kg/h (n = 13) respectively. Mean pH levels of the fetal and maternal effluent perfusates were 7.26 +/- 0.02 (n = 5) and 7.32 +/- 0.01 (n = 5). None of these parameters varied significantly from 1 to 4 h of perfusion. ACh output into the fetal vessels after 1 h of perfusion was 0.31 +/- 0.02 nnmol/min/g wt weight (n = 5) in the presence of physostigmine (2.7 uM) and did not vary significantly from 1 to 4 h of perfusion. Perfusion with Ca+(+)-free Krebs solution and 2 mM EDTA (but not with Krebs in the absence of EDTA, or with both Ca++ and EDTA, or in the presence of 3.78 mM Ca++) for 60 min resulted in a significant reduction of ACh output in the fetal perfusate. Output in the former case was partially restored on subsequent perfusion with normal Krebs for 60 min. These results suggest ACh release in perfused human placenta lobules is at least partially Ca+(+)-dependent.