To evaluate the frequency of anti-alpha-enolase antibodies in the sera of mothers whose children have congenital heart block (CHB), given provocative results in which alpha-enolase, a membrane protein, was recognized by monoclonal antibodies reactive with the peptide p200 of 52 kDa Ro/SSA in a neonatal rat heart library. An ELISA using a recombinant alpha-enolase protein was developed. Sera from 100 anti-Ro52+ CHB mothers in the Research Registry for Neonatal Lupus, 50 patients with systemic lupus erythematosus (SLE; 7 anti-Ro52+), and 48 healthy controls were tested for anti-alpha-enolase reactivity. There were no significant differences in the median values obtained from CHB mothers, patients with SLE, or controls at each of the dilutions tested. Only 7 (7%) at 1:100 dilution and 2 (2%) at 1:1000 dilution of 100 CHB sera were 3 standard deviations above the mean value obtained for controls. Preincubation with recombinant Ro52 did not inhibit anti-alpha-enolase reactivity. The low frequency of anti-alpha-enolase antibodies in the sera of CHB mothers and the absence of apparent cross-reactivity with Ro52 suggest that antibodies to Ro52 are not likely to mediate CHB via binding to alpha-enolase.