A general method has been developed for determining the number of mole
cules per asymmetric unit, Z(a), of a protein crystal. The method, vol
ume-specific amino acid analysis, relies on the direct measurement of
the number of protein molecules in a crystal as normalized by the crys
tal volume. The method is accurate, straightforward to implement and s
ensitive enough for application to small (0.002 m3) crystals. Although
the procedures described here are specific to proteins, extension to
nucleic acid crystals should be possible.