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Astroviruses are a new family of positive-stranded RNA viruses that cause gastroenteritis in a wide range of animals and in humans. Seven types of astrovirus, tentatively considered serotypes, have been distinguished by enzyme-linked immunosorbent assays (ELISA) or immunoelectron microscopy, but it is unclear whether the serotype designation is used properly. To test human sera for the presence of neutralizing antibodies and to type field strains, neutralization tests (NT) using CaCo2 tissue-culture-adapted astrovirus strains 1 to 7 and the corresponding rabbit reference sera were developed. In rabbits, neutralizing antibodies were predominantly serotype specific, with the exception of low-level cross-reactivity in astrovirus serotype 4 reference serum with astrovirus serotype 1 virus. Similarly, in humans, no evidence of cross-reactivity was found for the serotype combinations tested (all except the combination 1 and 7 and the combination 6 and 7). Typing by NT was concordant with typing by ELISA and genotyping, with one exception. The seroprevalence rates of neutralizing antibodies in an age-stratified sample of the population in Utrecht Province (n = 242) were 91% for astrovirus serotype 1, 69% for astrovirus serotype 3, 56% for astrovirus serotype 4, 36% for astrovirus serotype 5, 31% for astrovirus serotype 2, 16% for astrovirus serotype 6, and 10% for astrovirus serotype 7. Acquisition of antibodies was slower among persons seropositive for astrovirus serotype 5 than among those seropositive for astrovirus serotypes 1 to 4, suggesting that the epidemiology of serotype 5 astrovirus is different from that of astrovirus serotypes 1 to 4.