Protein kinases have been implicated in a number of regulatory mechanisms including signal transduction in many cells. To address the possibility that the large granular lymphocyte (LGL) also uses one or more unique protein kinases for LGL functions, an efficient method was developed to obtain partial cDNA clones for putative protein kinases from a rat LGL tumor cell line, RNK. Using the polymerase chain reaction (PCR), one hundred and nine amplified DNA segments were cloned and sequenced from a RNK cDNA library. One hundred and eight of these segments were putative protein kinases based on their deduced sequences. Among these were nine putative protein kinases, seven of which were putative tyrosine kinases and two were putative serine kinases. Only four of the putative kinases identified in this study were identical, or nearly identical, to previously published protein kinases. The deduced amino acid sequences of the remaining clones differed by seven or more amino acid residues in the amplified segments from all known protein kinase, and were considered to be novel putative protein kinases. Two of the five novel putative kinases showed lymphoid-restricted patterns of expression on Northern analyses. The method described in this paper provides an efficient cloning strategy for novel protein kinases, and is similar to that published in a previous report. Although the PCR primers used in this report differ only slightly from those in the previous report, we used a much higher annealing temp (50 degrees C) than in the previous report (37 degrees C), which may in part account for our higher yield of putative protein kinase sequences.