Water soluble extracts of 3131 adult specimens of Dicrocoelium dendriticum from cattle, sheep and goats, mainly from León province, were analysed by isoelectric focusing in thin-layer polyacrylamide gels. Activity of the following enzymes was studied: lactate dehydrogenase (LDH, EC 126.96.36.199), glucose phosphate isomerase (GPI, EC 188.8.131.52), phosphoglucomutase (PGM, EC 184.108.40.206), acid phosphatase (AcP, EC 220.127.116.11), α-glycerophosphate dehydrogenase (α-GPDH, EC 18.104.22.168), hydroxybutyrate dehydrogenase (HBDH, EC 22.214.171.124) and malate dehydrogenase (MDH, 126.96.36.199). Five distinct enzyme types were recognized for LDH (pH range 6.30–7.13), GPI (pH 6.13–6.80) and PGM (pH 6.20–6.60) whereas AcP showed three different patterns (pH 5.70–5.92). Weak and diffuse activity was detected for MDH (pH 4.8–6.2) and no activity was observed for α-GPDH and HBDH. In general, little phenotypic variation was observed between worms recovered from a single host, between those from hosts of the same species and between those from hosts of different species, although some enzyme types were found in some animals but not others. Nevertheless, it must be taken into account that most parasites came from sheep and also from a relatively small area in north-west Spain.