Objective: The aim was to study pathways of nucleotide catabolism and adenosine production in cultured human umbilical vein endothelial cells (HUVEC) incubated under normal conditions and following inhibition of ATP synthesis. Methods: Confluent cultures of HUVEC were incubated for 45 min in Hank's balanced salt solution in a 95%O2/5%CO2 atmosphere under the following experimental conditions: (1) in presence of the adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA); (2) with EHNA and the adenosine kinase inhibitor, 5'-iodotubercidin (ITu); (3) with iodoacetate and oligomycin (I + O), inhibitors of ATP production, and EHNA; (4) with I + O, EHNA, and an inhibitor of ecto 5'-nucleotidase, α,β-methyleneadenosine 5'-diphosphate (AOPCP). Nucleotide and catabolite contents in both cells and medium were analysed by HPLC. Results: The initial contents of ATP, ADP, and AMP were 9.12(SEM 1.2), 0.73(0.08) and 0.11(0.02) nmol per culture flask respectively. These levels were maintained under experimental condition (1), with only a small increase in hypoxanthine in the medium of 0.9(0.2) nmol. Under experimental condition (2), adenosine accumulation in the medium was greatly enhanced [increase by 0.9(0.2) nmol], while the rise in hypoxanthine was similar to that in experimental condition (1). Under experimental condition (3), cellular ATP was totally depleted after 45 min and AMP increased to 4.3(0.9) nmol. Adenosine in the medium was increased by 5.4(0.2) nmol, the increase in the sum of hypoxanthine and inosine was 1.6(0.2) nmol, and the increases in uric acid and xanthine were less than 0.4 nmol. Under experimental condition (4), where AOPCP was present in addition to I + O and EHNA, adenosine production was markedly reduced and AMP accumulated in the medium [1.2(0.2) nmol]. Conclusions: Adenosine is generated continuously in cultured HUVEC under normal conditions, but is immediately recycled via adenosine kinase. In ATP depleted cells, nucleotide catabolism proceeds predominantly via intracellular dephosphorylation of AMP with a small contribution from the extracellular dephosphorylation pathway. The capacity of the AMP deamination pathway in endothelium is small and the flux through xanthine oxidoreductase is minimal. Cardiovascular Research 1994;28:100-104