We have identified a new cyclic-nucleotide phosphodiesterase isoform, PDE3A, and cloned its cDNA from cultured aortic myocytes. The nucleotide sequence of its coding region is similar to that of the previously cloned myocardial isoform except for the absence of the initial 300–400nt that are present in the latter, as confirmed by reverse-transcriptase-mediated PCR, 5′ rapid amplification of cDNA ends and a ribonuclease protection assay. Expression in Spodoptera frugiperda (Sf9) cells yields a protein with catalytic activity and inhibitor sensitivity typical of the PDE3 family. The recombinant protein's molecular mass of approx. 131kDa is compatible with translation from an ATG sequence corresponding to nt 436–438 of the myocardial PDE3A coding region. Antibodies against residues 424–460 (nt 1270–1380) and 1125–1141 (nt 3373–3423) of the myocardial isoform react with an approx. 118kDa band in Western blots of homogenates of human aortic myocytes, whereas antibodies against residues 29–42 (nt 85–126) do not react with any bands in these homogenates. Our results suggest that a vascular smooth-muscle isoform (‘PDE3A2’) is a product of the same gene as the longer myocardial (‘PDE3A1’) and the shorter placental (‘PDE3A3’) isoforms and is generated pre-translationally in a manner that results in the absence of the 145 N-terminal amino acids of PDE3A1.