Three in vitro cytotoxicity assays [neutral red uptake assay (NRU), MTT test and total protein content determination (TPC)] were analysed to assess their value for predicting the ocular irritancy potential of 20 surfactants. For each test, three established cell lines (SIRC rabbit corneal cells, Balb/c 3T3 and L929 mouse fibroblasts) were used. The concentration that induced 50% inhibition relative to controls (IC(50)) was calculated for each test, cell line and chemical. In vivo ocular irritancy data were compared with in vitro results. None of these assays provided a marked correlation of surfactant ocular irritation (Spearman or Pearson correlation coefficient lower than 0.65, P < 0.01 and moderate agreement with Kappa test). An IC(50) threshold of 700 mug/ml was set to discriminate between surfactant irritation and non-irritation. Three compounds were detected as false negatives (CHAPS, CHAPSO and sodium taurocholate), and two as false positives (Triton X155 and Brij 35).