Bacteriophage T4 serine and proline transfer RNAs are derived from a common precursor RNA. This precusor RNA lacks -C-C-A sequences which could provide 3' termini for the mature transfer RNAs. We have deduced part of the pathway leading to the formation of the C-C-A sequences in the transfer RNAs by characterizing incompletely matured precursor molecules which accumulate during infection of mutant hosts that lack specific enzymes associated with transfer RNA metabolism. Maturation is initiated by the addition of -C-C-AOH to the 3' terminus of the precusor RNA through the combined actionof an unidentified nuclease and tRNA nucleotidyltransferase (EC 126.96.36.199). Precursor RNA molecules terminating in -C-C-AOH is serine transfer RNA and the second product is immature proline transfer RNA. The terminal steps leading to proline transfer RNA have not been fully delineated, but are known to involve the replacement of a -C-UOH sequence by -C-C-AOH.