The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into alpha and beta subunits under suitable conditions of polyacrylamide gel electrophoreis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin, and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tublin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells from flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.