We have studied the phenotype of a heat-sensitive mutation that defines lambda tail gene T. Induction and growth at the nonpermissive temperature of a lambda Tts40 prophage results in production of morphologically normal but biologically inactive tails, which can be activated in vitro by lysates supplying the products of genes T, U, and Z. Thus, gene T acts between genes V and U in the tail assembly pathway or immediately after the completion of tail shaft polymerization. lambda Tts40 lysates also contain a substantial number of morphologically normal phage particles which are deficient in cleavage of the minor tail protein gpH. This result is consistent with the fact that gpH cleavage normally occurs later in the assembly pathway than the time found for gene T action. Purified lambda Tts40 virions that were produced at the permissive temperature are normal with respect to gpH cleavage. However, they are more sensitive to heat inactivation than are wild-type virions, suggesting that the T gene product is in the virion. At the permissive temperature, the mutant protein made by lambda Tts40 is active in vivo even if it has been synthesized at the nonpermissive temperature.