Syncytiotrophoblast, the transport epithelium of the human placenta, is maintained during pregnancy by cellular turnover, involving apoptosis and loss of syncytial nuclei which are replaced by differentiation and fusion of cytotrophoblast cells (1). In non-placental tissues, voltage-gated K⁺ (K_v) channels participate in cellular fusion and developmental regulation (2,3). The placenta expresses mRNA for K_v channel subtypes (4) but their role in syncytiotrophoblast development remains to be explored. The aim of this study was to determine the effect of 4-aminopyridine (4-AP), a blocker of K_v channels, on cytotrophoblast differentiation using a well characterised in vitro model. Human cytotrophoblast cells were isolated from 6 normal term placentas and maintained in culture for 66h as previously described (5). At 18h, cells are mononucleate and by 66h they aggregate, fuse and differentiate into multinucleate syncytia. An increase in human chorionic gonadotrophin (hCG) secretion accompanies morphological differentiation (5). Cells were exposed to 0.01-5.0mM 4-AP over 18-66h or left untreated. Culture medium was collected at 18 and 66h, and analysed for hCG and lactate dehydrogenase (LDH) activity, markers of differentiation and cell death respectively. Cell protein was determined at 18 and 66h and cells fixed in methanol for qualitative assessment of aggregation and multinucleation by phase contrast microscopy. Paired 18 and 66h untreated controls were analysed using a Mann Whitney U test. The effects of 4-AP at 66h were expressed as a % of the corresponding 66h control (100%) and the median and interquartile range compared to 100% using a Wilcoxon Signed Rank Test. In control cultures, mononucleate cells at 18h aggregated and fused to become multinucleated by 66h. This was associated with an increase in hCG secretion from 7 to 150 mIU/ml/mg protein/h (18 vs 66h: p<0.02) and a fall in LDH release from 0.7 to 0.2 absorbance units/mg protein/h (18 vs 66h: p<0.03). At 0.1 and 0.01mM, 4-AP significantly inhibited hCG secretion to 86% (75-92%; p<0.04) and 72% (46-93%; p<0.02) of control, without any affect on either cell protein or LDH release; microscopic examination revealed cell aggregation but multinucleation was less evident than with 66hr controls. 5.0mM 4-AP reduced hCG secretion and cell protein at 66h to 0.9% (0-21%; p<0.02) and 34% (16-43%; p<0.02) of control and simultaneously increased LDH release to 361% (267-761%; p<0.02) of control. Extensive cellular necrosis was observed indicating that 4-AP was cytotoxic at 5.0mM. Inhibition of hCG secretion by 0.01 and 0.1mM 4-AP implicates a role for K_v channels in cytotrophoblast differentiation. Further work is needed to characterise K_v channel participation in maintenance of syncytiotrophoblast. In summary, we demonstrated that hCG secretion was inhibited by 0.01 and 0.1mM 4-AP, suggesting that Kv channels participate in cytotrophoblast cell differentiation in vitro.