Here, we tested the hypothesis that a promiscuous bacterial cyclase synthesizes purine and pyrimidine cyclic nucleotides in the pulmonary endothelium. To test this hypothesis, pulmonary endothelial cells were infected with a strain of the Gram-negative bacterium Pseudomonas aeruginosa that introduces only exoenzyme Y (PA103 exoUexoT::Tc pUCPexoY; ExoY⁺) via a type III secretion system. Purine and pyrimidine cyclic nucleotides were simultaneously detected using mass spectrometry. Pulmonary artery (PAECs) and pulmonary microvascular endothelial cells (PMVECs) both possess basal levels of four different cyclic nucleotides in the following rank order: cAMP > cUMP cGMP cCMP. Endothelial gap formation was induced in a time-dependent manner following ExoY⁺ intoxication. In PAECs, intercellular gaps formed within two hours and progressively increased in size up to six hours, when the experiment was terminated. cGMP concentrations increased within one hour post-infection, while cAMP and cUMP concentrations increased within three hours, and cCMP concentrations increased within four hours post-infection. In PMVECs, intercellular gaps did not form until four hours post-infection. Only cGMP and cUMP concentrations increased at three and six hours post-infection, respectively. PAECs generated higher cyclic nucleotide levels than PMVECs, and the cyclic nucleotide levels increased earlier in response to ExoY⁺ intoxication. Heterogeneity of the cyclic nucleotide signature in response to P. aeruginosa infection exists between PAECs and PMVECs, suggesting the intracellular milieu in PAECs is more conducive to cNMP generation.