Background Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit [alpha].sub.1G may be a fetal target of maternal sera autoantibodies in CHB. Methodology/Principal Findings We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G ([alpha].sub.1G gene) in the AV junction of human fetal hearts compared to the apex (18-22.6 weeks gestation). Using human fetal hearts (20-22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the [alpha].sub.1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the [alpha].sub.1G protein. By ELISA we demonstrated maternal sera reactivity to [alpha].sub.1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305-319 of the extracellular loop linking transmembrane segments S5-S6 in [alpha].sub.1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the [alpha].sub.1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells. Conclusions/Significance Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of [alpha].sub.1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for [alpha].sub.1H suggesting that autoantibodies can target multiple fetal targets.