We studied the ontogeny of concentration and in vitro phosphorylation of an 85 kDa Triton-insoluble protein from cerebral cortex of 7, 15, 21 and 90 day old rats. The Triton-insoluble cytoskeletal fraction contains an 85 kDa basic phosphoprotein different from synapsin 1, as determined by nonequilibrium pH gradient electrophoresis and phosphopeptide mapping with V8 protease. The concentration of the 85 kDa cytoskeletal associated phosphoprotein was analyzed during development. Results indicated that the concentration of this protein oscillated during suckling, presenting a maximal value at day 15 and decreasing again to stabilize at values near those of 7 day old rats, remaining constant in 21 and 90 day old animals. However, in vitro 32P incorporation, expressed as cpm/microgram, presented a developmentally regulated pattern, with maximal values in young rats, declining with age to negligible values in 90 day old animals. The endogenous phosphorylating system responsible for in vitro 32P incorporation into the 85 kDa protein was determined by the addition of specific activators of second-messenger protein kinases (cAMP, Ca2+/ calmodulin and Ca2+/phosphatidylserine/phorbol ester) and a protein phosphatase inhibitor (okadaic acid) to the incubation system. Results suggested that the in vitro phosphorylation system is composed of protein kinase A, Ca2+/calmodulin dependent protein kinase and protein phosphatase 1.