Microtubule-associated proteins (MAP) have been identified in cultures of rat sympathetic neurons. In all of the experiments performed here, the cultures consisted of greater than 97% neurons. 26 proteins were identified in these neuronal cultures that (a) remained associated with cytoskeletons prepared with a Triton X-100-containing microtubule-stabilizing buffer, (b) were released from such cytoskeletons by incubation in microtubule-depolymerizing buffers, (c) were not detected in cytoskeletons prepared from cultures depleted of microtubules by treatment with podophyllotoxin, and (d) co-cycled with rat brain microtubule proteins. We conclude that these 26 proteins are associated with microtubules in sympathetic neurons. Two of these proteins have molecular weights of approximately 30,000 and isoelectric points of approximately 6.2; the rest of the proteins range in molecular weight from 60,000 to 76,000 and isoelectric point from 6.3 to 6.9. This latter group of MAPs was heat labile. Several other proteins in the neuronal cultures had the solubility properties and drug-lability expected of MAP. All of these proteins had apparent molecular weights greater than 200,000; one of these putative MAP co-migrated with rat brain MAP-1. We did not detect any putative MAP in these cultures that co-migrated with rat brain MAP-2. In isoelectric focusing-SDS PAGE, the 24 MAP with molecular weights of 60,000-76,000 appeared to comprise four distinct molecular weight classes. Each molecular weight class was in turn composed of several proteins that varied in isoelectric point. In peptide mapping experiments, the isoelectric variants of each molecular weight class gave rise to very similar peptide maps. These observations suggest that each molecular weight class consists of several closely related proteins. It was also determined that all except the most basic member of the four MAP classes could be phosphorylated in vivo, raising the possibility that differential phosphorylation contributed to the variation in the isoelectric points of the members of each MAP class. We performed pulse-chase experiments to further evaluate the contribution of posttranslational modification to the generation of the complex population of MAP in the molecular weight range of 60,000 to 76,000. In cultures labeled for 20 min, only the more basic members of each MAP class were detectably labeled, while in cultures labeled for 20 min and then chased for 220 min the more acidic members of the MAP classes became labeled.(ABSTRACT TRUNCATED AT 400 WORDS)