To determine the role of the FHIT tumor suppressor gene product, a diadenosine 5',5'''-P1,P3-triphosphate (Ap3A) hydrolase, in the regulation of the concentration of Ap3A and diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) in vivo, the levels of the adenosine(5')triphospho(5')nucleoside (Ap3N) and adenosine(5')tetraphospho(5')nucleoside (Ap4N) families were measured by luminometry in a number of human cell lines and correlated with the expression of Fhit determined by immunoblotting. Fhit-positive cells had no Ap3N or a very low level of Ap3N, whereas most Fhit-negative cells had Ap3N in the range 0.2-0.9 pmol/10(6) cells. Ap4N (mean value, 0.17 pmol/10(6) cells) did not correlate with Fhit expression. The results suggest that Fhit efficiently metabolizes Ap3A and Ap3N but not Ap4A or Ap4N in vivo.