Changes in the cellular content of cyclic AMP and in the activities of adenylyl cyclase, cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinases during differentiation of rabbit bone marrow erythroid cells were investigated. The cells were separated by velocity sedimentation at unit gravity into six fractions corresponding to different stages of development: proerythroblasts, basophilic erythroblasts, polychromatic cells, early orthochromatic and late orthochromatic cells and reticulocytes. Adenylyl cyclase activity was found to decrease continuously as the cells developed, from approx. 180 pmoles cyclic AMP formed/mg of protein/20 min in proerythroblasts to 10 pmoles in circulating reticulocytes. The proerythroblasts were the richest cells in cyclic AMP which is present at a cellular concentration of approx. 1.4 microM. In basophilic cells the cyclic AMP content was about 80% lower than in proerythroblasts. No further changes in cyclic AMP levels were observed after the final cell division. Cyclic AMP phosphodiesterase was found to be very active in the most immature cells, the proerythroblasts. After differentiation into basophilic erythroblasts, a 4-fold decrease in cyclic AMP phosphodiesterase activity occurred. In polychromatic cells there was a further drop in phosphodiesterase activity and after the last cell division the enzyme activity was constant and very low. Both cytosolic cyclic AMP-binding capacity and cytosolic cyclic AMP-dependent protein kinase activity decreased in dividing rabbit bone marrow erythroblasts when calculated in terms of cell number but remained constant per cell volume. After the final cell division, cyclic AMP-dependent protein kinase activity did not change further, whereas cyclic AMP-binding capacity declined. There were no qualitative but only quantitative changes in the cyclic AMP-binding proteins that are present in the cytosol of developing erythroblasts. In the immature cells, the apparent Kd for the interaction of binding proteins with cyclic AMP was 4 . 10(-8) M. The data suggest that changes in cyclic AMP-binding activity during differentiation of erythroid cells are due both to changes in the amount of binding proteins and their affinity for cyclic AMP. The phosphorylation of rabbit erythroblast plasma membrane proteins by membrane-associated protein kinase(s) was found to be cyclic AMP-dependent in dividing cells during the early stages of differentiation. When the erythroid cells reach the non-dividing stage in their development, autophosphorylation of membrane ghosts was no longer stimulated by cyclic AMP.(ABSTRACT TRUNCATED AT 400 WORDS)