We have attempted a biochemical characterization of the microtubule-associated proteins (MAPs) of cultured HeLa cells. The HeLa MAPs consist of a group of three polypeptides of 200,000 to 220,000 molecular weight (the 210K MAP) and a protein of 125,000 molecular weight (the 125K MAP). The solution properties of the HeLa MAPs were examined using molecular sieve chromatography and sucrose gradient sedimentation. With both analytical procedures, the 125K and 210K MAPs behaved independently of one another. The effects of each of the MAPs on microtubule polymerization were also studied. Both the 125K and 210K MAPs stimulated the polymerization of pure tubulin. The effect of high levels of MAPs on microtubule polymerization was also examined. Increasing the concentration of MAPs at a constant tubulin concentration increased both the rate and extent of microtubule polymerization. The 125K and 210K MAPs showed independent behavior with respect to binding to microtubules. The 210K MAP saturated its binding sites at a level of 14.0% (210K MAP:tubulin in polymer, w/w), while the 125K MAP showed no saturation even at a level of 18.3%. These results demonstrate that the 125K and 210K MAPs are distinct molecular species, differing in their solution properties and their binding to microtubules. The 210K MAP showed some similarities and some differences when compared to the porcine brain high molecular weight MAP. The HeLa MAPs, although showing some properties similar to brain MAPs, are nevertheless distinctive in several respects and may best be considered as separate though possibly related species.