Neurofilament preparations isolated from bovine spinal cord contain cyclic-AMP-dependent protein kinase (PKA) activity. Treatment of this preparation with cyclic AMP, to dissociate the regulatory subunit of the kinase from the catalytic subunit, resulted in retention of the kinase activity but loss of cyclic AMP regulation. This suggests that PKA is associated via its catalytic subunit with the neurofilament preparation. The association of exogenous PKA from bovine heart with the neurofilament preparation and with neurofilaments reconstituted from purified neurofilament proteins was also investigated. Either the free catalytic subunit or combinations of the catalytic and regulatory subunits of PKA were incubated with the preparations, and the degree of association was determined as the level of kinase activity that co-sediments with neurofilaments. The results indicate that the free catalytic subunit of PKA co-sediments with neurofilaments reconstituted from purified proteins. The regulatory subunit of PKA from bovine heart, when pre-mixed with the catalytic subunit, decreased the level of kinase that co-sediments with the neurofilament fraction in a dose-dependent manner. This effect of the regulatory subunit was reversed by inclusion of cyclic AMP in the incubation medium before centrifugation. The above findings suggest that the regulatory subunit, when attached to the catalytic subunit, has an inhibitory effect on its association with neurofilaments, with the implication that the association may be a cyclic-AMP-regulated event.