It was recently reported that sera from patients with systemic lupus erythematosus contain antibodies reactive with the second extracellular loop of the serotoninergic 5-HT₄ receptor expressed in the human heart. This antibody response was associated with antibodies to 52kD SSA/Ro, a reactivity prevalent in mothers of children with congenital heart block (CHB). The current study was undertaken to determine whether the 5-HT₄ receptor is a target of the immune response in these mothers. Initial experiments demonstrated mRNA expression of the 5-HT₄ receptor in the human foetal atrium. Electrophysiologic studies established that human foetal atrial cells express functional 5-HT₄ receptors. Sera from 116 mothers enrolled in the Research Registry for Neonatal Lupus, whose children have CHB, were evaluated. Ninety-nine (85%) of these maternal sera contained antibodies to SSA/Ro, 84% of which were reactive with the 52kD SSA/Ro component by immunoblot. None of the 116 sera were reactive with the peptide spanning aa165–185 of the serotoninergic receptor. Rabbit antisera which recognized this peptide did not react with 52kD SSA/Ro or peptide aa365–382 in the C terminus. Although 5-HT₄ receptors are present and functional in the human foetal heart, maternal antibodies to the 5-HT₄ receptor are not associated with the development of CHB.