The expression of the class III β‐tubulin isotype was studied in cultured brain neurons by means of a monoclonal antibody (TuJ1). The results obtained indicate that during early axonal outgrowth most of the class III β‐tubulin is not incorporated into microtubules, a phenomenon which is also observed under conditions which alter the rate and extent of the neurite outgrowth response. On the other hand, a dramatic increase in its incorporation into microtubules is observed after the neurons have differentiated their neurites as axons and dendrites. In addition, the appearance of colchicine‐resistant microtubules containing this isotype, a phenomenon which occurs late in neurite development, is highly coincident with the appearance of stable microtubules containing high molecular weight microtubule‐associated proteins (MAPs). This pattern is different from that of the accumulation and incorporation of other β‐tubulin isotypes into microtubules. Taken collectively, our results indicate that differences exist in the in vivo utilization of tubulin isotypes in developing brain neurons and suggest that the class III β‐tubulin isotype is not a primary factor involved in the regulation of microtubule assembly during early neurite out‐growth, but that it may be important for maintaining further neurite elongation and/or determining some unique binding property of MAPs to specific microtubule subsets. © 1992 Wiley‐Liss, Inc.