To better understand the function(s) of the multiphosphorylation repeat (MPR) of the high molecular weight neurofilament (NF) subunit (NF-H), we sought to determine how phosphorylation within this region is regulated in cultured rat sympathetic neurons. To do this, monoclonal antibodies specific to phosphorylated or nonphosphorylated tandem repeats of the amino acid sequence Lys-Ser-Pro-Ala-Glu-Ala found within the MPR were identified and used to determine the extent of phosphorylation, the time course of phosphorylation, and the rate of turnover of phosphate groups within the NF-H MPR in cultured sympathetic neurons. We showed that (1) the synthesis and phosphorylation of NF-H occurs in these neurons cultured for 1 or 4 weeks; (2) the conversion from poorly to more highly phosphorylated variants of NF-H occurs slowly in cultured neurons; and (3) the turnover of phosphate groups on both poorly and highly phosphorylated variants of NF-H occurs more rapidly than the turnover of NF-H itself. In addition, we showed that the 200-kD highly phosphorylated NF-H can contain at least five consecutive nonphosphorylated tandem repeats as well as phosphorylated tandem repeats, whereas we were unable to detect consecutively phosphorylated tandem repeats in the 160-kD form of NF-H. These findings allow us to propose a mechanism whereby NF-H is initially phosphorylated singly at sites distributed throughout the MPR. This "poorly phosphorylated NF-H," which is the predominant form in immature neurons, is then converted by phosphorylation at additional sites within the MPR to the more "highly phosphorylated" species of NF-H typically found in mature neurons.(ABSTRACT TRUNCATED AT 250 WORDS)