Pyruvate kinase (PK) has four isozymes (L, R, M1, M2) that are encoded by two different genes of PK L and M. Differential splicing produces L- and R-type PK mRNA and M1- and M2-type PK mRNA from the PK L gene and the PK M gene, respectively. The nucleotide sequences of the 3'-noncoding region are the same between the L- and the R-type PK and between the M1- and the M2-type PK. We isolated 3'-noncoding sequences of human L- and M2-type PK cDNA to construct L-type and M-type PK specific probes. With these probes, we performed Northern blot analysis of the RNA samples extracted from human tissues. Northern blot analysis showed that both kidney and liver had mRNAs that hybridized with both the L and M probes. Small intestine, skeletal muscle, brain, testis, and lung mRNAs hybridized only with the M probe. Our probes are considered useful for the detection of the types of PK isozymes expressed in small amounts, which are very difficult to detect using the conventional PK polyacrylamide gel electrophoretic method.