SummaryThin layer agarose isoelectric focusing (IEF) was used to separate proteins from the larvae ofTipula oleraceaLinnaeus andTipula paludosaMeigen (Diptera, Nematocera). Silver staining revealed protein banding patterns which consistently distinguished these two species at any stage in their life cycles. Within the pH range 5–6Tipula paludosacontained one major protein band andT. oleraceatwo bands, with the pI value of theT. paludosaprotein being slightly higher than those ofT. oleracea. The IEF method is particularly useful for the larval stages of these species because they are often visually inseparable. Leather jackets obtained during a survey of Northern Ireland and north‐eastern and western Scotland were tested to determine the distribution and frequency of these species occurring in grassland. In each area surveyedT. paludosapredominated with only one field in Northern Ireland and north‐eastern Scotland found to containT. oleracea. In western ScotlandT. oleraceaoccurred in five fields each year of the survey, and was locally abundant on the Isle of Bute in the 1990/91 survey making up 15% of the total larvae t