Two isoforms of the protein kinase A catalytic subunit, C alpha and C beta, have previously been described in the mouse. We now report the cloning and characterization of a novel C-related sequence, Cx, from a murine genomic library. Cx is 89.8% identical to part of the C alpha coding region, but lacks all of the introns present in this gene, suggesting that it arose via retroposition. The existence of several frameshift mutations, premature termination codons, and missense mutations at critical sites confirms that it is a pseudogene. Furthermore, we are unable to detect any expression. Homology with functional protein kinase genes commences exactly at the first intron splice junction in C alpha, downstream of the expected translational start codon. Cx is also truncated at its 3' end by the interposition of two distinct, contiguous LINE-1 elements. By fluorescence in situ hybridization, we demonstrate that Cx is located on the X Chromosome (Chr), at band F3. This is displaced from its functional homologs, C alpha and C beta, which we map to mouse Chrs 8 (band C3) and 3 (band H3), respectively.