The development in primary dissociated rat brain cultures of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity, the accumulation of CNP protein, and the number of cells accumulating this protein have been quantitatively determined as a function of time in culture. Parallel determinations have been made for the first two parameters for developing rat brain. The developmental profile of CNP enzymatic activity and amount of CNP protein in culture paralleled that observed in rat brain, in which the period of most active development occurred 7-25 days after birth. Mean CNP activities of 5.6 and 8.1 pmol/min/rng total protein were recorded for the cultures and rat brain, respectively, at their maximal levels. The corresponding mean values for the CNP protein accumulation were calculated to be 138 and 150 prnolimg total protein, respectively. Thus maximal specific activities of the CNP protcin were estimated to be about 800 and 1,100 pmollmini mg CNP protein for culture and rat brain enzyme, respectively. Approximately three million cells expressing CNP appeared in the cultures per dissociated fetal rat brain seeded. Each CNP+ oligodendroeyte in culture had an average CNP activity of 3.2 pmolhin, and an average CNP protein content of 0.09 fmol (5.4 x lo7 molecules), values which remained nearly constant during the course of development. Two principal conclusions are drawn from these data. First, the dissociated fetal brain culture system reproduces rather accurately the tempord developmental pattern of CNP expression occurring in the rat brain, but some important quantitative differenceb occur which suggest the need for additional environmental stimuli missing in these cultures. Second, the quantitative increases in CNP specific activity and amount of CNP protein occurring during oligodendrocyte differentiation in these cultures are primarily the result of increases in the number of CNP+ cells present which upon differentiation express very quickly, via an off-on regulation, steady-state levels of the enzyme.