Abstract A reverse-phase high-performance liquid chromatographic (h.p.l.c.) system is developed for a rapid (≈ 20 min) separation and quantification of fourteen chlorophylls and their breakdown products and seventeen carotenoids from acetone extracts of algal cultures and natural waters. An ion-pairing reagent is included to achieve good resolution with the acidic chloropigments (chlorophyllides and phaeophorbides). Fluorescence and absorbance detectors are used to quantify chloropigments and carotenoids respectively, with detection limits of 0.01–0.2 ng for these pigments and 200–600 ng for carotenoids. Chlorophyll a at concentrations of 0.1 ng l −1 may be detected in sea water. Methanol is shown to be unsuitable for extracting pigments because it causes allomerization and transesterification of chlorophylls and chlorophyllides. The spectrophotometric determination of chlorophyll pigments in samples containing breakdown products grossly overestimates the actual concentration of these pigments, as determined by h.p.l.c.