Dynactin is a multiprotein complex that enhances dynein activity. The largest dynactin subunit, p150Glued, interacts with microtubules through its N‐terminal region that contains a globular cytoskeleton‐associated protein (CAP)‐Gly domain and basic microtubule‐binding domain of unknown structure. The p150Glued gene has a complicated intron–exon structure, and many splice isoforms of p150Glued protein have been predicted. Here we describe novel natural 150 kDa isoforms: the p150Glued‐1A isoform, whose basic domain is composed of 41 amino acids, and p150Glued‐1B with a basic domain of 21 aa because of the lack of exons 5–7 in the corresponding messenger RNA (mRNA). According to reverse transcriptase‐polymerase chain reaction (RT‐PCR) and western blot data, p150Glued‐1A is expressed in nerve tissues, in cultured cells and in embryonic tissues, while 1B is expressed ubiquitously. Overexpression of GFP‐p150Glued‐1A and ‐1B fusion proteins and immunostaining of cultured cells with 1A‐specific antibodies show that the p150Glued‐1A isoform is distributed along microtubules, whereas 1B is associated with microtubule plus‐ends. The higher affinity of the p150Glued‐1A isoform for microtubules is confirmed by a co‐pelleting assay. In fibroblast‐like cells, the interaction of p150Glued‐1A with microtubules is less dependent on EB1/EB3 and CLIP170 proteins, compared with p150Glued‐1B. In polarized cells, p150Glued‐1A decorates microtubules that face the leading edge of the cell. The pattern of p150Glued‐1A and p150Glued‐1B interaction with microtubules and their tissue‐specific expression patterns suggest that these isoforms might be involved in cell differentiation and proliferation.