Lens epithelial cell proliferation is regulated by growth factors in the aqueous humour of the eye. Although the lens fibre cell-differentiating factors are well defined, the factors in aqueous that promote lens cell proliferation are not. Mitogens present in aqueous primarily signal through the MAPK/ERK and PI3-K/Akt pathways. By characterising the signalling pathways involved in lens cell proliferation, we aim to identify the factors in aqueous that regulate this process in vivo. Using rat lens epithelial explants, 5'-2'-bromo-deoxyuridine and H(3)-thymidine incorporation were used to compare the effects of aqueous, insulin-like growth factor (IGF-1), platelet-derived growth factor (PDGF-A), epidermal growth factor (EGF) and fibroblast growth factor (FGF-2) on lens cell proliferation. Western blotting was employed to characterise ERK1/2 and Akt signalling induced by these mitogens. The above assays were also repeated in the presence of selective receptor inhibitors. Similar to aqueous, FGF induced a sustained ERK1/2 signalling profile (up to 6 h), unlike IGF, PDGF and EGF that induced a transient activation of ERK1/2. In the presence of a FGF receptor (FGFR) inhibitor, the sustained aqueous-induced ERK1/2 signalling profile was perturbed, resembling the transient IGF-, PDGF- or EGF-induced profile. In the presence of other growth factor receptor inhibitors, aqueous maintained its sustained, 6 h, ERK1/2 signalling profile, although ERK1/2 phosphorylation at earlier time periods was reduced. No one-specific receptor inhibitor could block aqueous-induced lens cell proliferation; however, combinations of inhibitors could, providing FGFR signalling was blocked. Multiple growth factors are likely to regulate lens cell proliferation in vivo, with a key role for FGF in aqueous-induced signalling and lens cell proliferation.