Amebae of Naegleria gruberi differentiate into flagellates when transferred from growth medium to nonnutrient buffer. Experiments were performed to determine whether the tubulin that forms the flagellar microtubules pre-exists in amebae or is synthesized during differentiation. Amebae prelabeled uniformly with [(35)S]methionine were allowed to differentiate in the presence and in the absence of exogenous unlabeled methionine. In the presence of unlabeled methionine the flagellar tubulin contained only 30% as much [(35)S]methionine as in its absence. Thus at least 70% of the tubulin was synthesized de novo. Isotope dilution and pulse experiments showed that flagellar tubulin synthesis began one-third of the way through differentiation, before any morphological change had occurred. Flagellar tubulin antigen, as measured using a specific antiserum, also began to increase one-third of the way through differentiation and increased 35- to 55-fold during the course of differentiation. These experiments demonstrate that most if not all of the flagellar tubulin is synthesized de novo during differentiation.