Lowering the proportion of acetic acid in the standard 1:3 acetic acid:methanol chromosome fixative used both during initial fixation and subsequent washing produced up to a 20-fold increase in the yield of intact metaphases from cultures of several permanent cell lines. Although this inhibited chromosome spreading, addition of various acetic acid-methanol mixtures immediately after the cell suspension was dropped onto slides increased the degree of spreading and resulted in well-spread, cytoplasm-free metaphases.