A microparticle-enhanced nephelometric immunoassay was developed for alpha-lactalbumin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immunoagglutination of a microparticle-alpha-lactalbumin conjugate with an anti-alpha-lactalbumin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 1.5 micrograms/L) and could be performed in high dilution of milk, excluding any interference or sample pretreatment. It allowed the quantification of alpha-lactalbumin on a large range of concentrations (0.5-16.9 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 1 to 7%). Changes in the alpha-lactalbumin concentration of human milk during lactation were determined in 162 samples. The concentration and ratio of alpha-lactalbumin total protein were found to be significantly lower in colostrum (4.9 g/l, 27%) than in transitional milk (5.2 g/L, 40%), then decreased in mature milk (3.4 g/L, 31%).