A stable isotope dilution assay for galactitol in amniotic fluid has been developed using selected ion monitoring chemical ionization gas chromatography-mass spectrometry of the hexaacetate derivative. [1,1-2H2]Galactitol was synthesized for use as the internal standard. Galactitol is a component of normal amniotic fluid with a mean concentration of 0.70 +/- 0.18 mumol/liter (n = 5). The amniotic fluid of a fetus with galactosemia had a concentration of 7.96 mumol/liter. Mannitol, sorbitol, and inositol were also found to be normal constituents of amniotic fluid. This stable isotope dilution assay is a rapid accurate method for measurement of galactitol in amniotic fluid for prenatal diagnosis of galactosemia.