Procedures are described for studying protein phosphorylation in 1 mm diameter micro-slices of rat brain tissue using two-dimensional electrophoresis as analytical tool. The activity of several protein phosphorylating systems, including a major system phosphorylating a 40 kDa substrate complex, was highly dependent on the procedures used for micro-slice preparation and on the Ca2+-content of the preparation medium. Under optimal conditions the pattern of phosphorylation observed in micro-slices closely resembled that obtained by in vivo labelling.