Many nucleoside analog drugs, such as ribavirin and viramidine, are activated or metabolized in vivo through 5′-phosphorylation. In this report, we determined the steady-state kinetic parameters for 5′-monophosphorylation of ribavirin and viramidine by adenosine kinase. The apparent K_m for ribavirin is 540 μM, and k_cat is 1.8 min⁻¹. Its catalytic efficiency of 3.3 × 10⁻³ min⁻¹ · μM⁻¹ is 1,200-fold lower than that of adenosine. In contrast to the common belief that ribavirin is exclusively phosphorylated by adenosine kinase, cytosolic 5′-nucleotidase II was found to catalyze ribavirin phosphorylation in vitro. The reaction is optimally stimulated by the physiological concentration of ATP or 2,3-bisphosphoglycerate. In phosphate-buffered saline plus ATP and 2,3-bisphosphoglycerate, the apparent K_m for ribavirin is 88 μM, and k_cat is 4.0 min⁻¹. These findings suggest that cytosolic 5′-nucleotidase II may be involved in ribavirin phosphorylation in vivo. Like ribavirin, viramidine was found to be phosphorylated by either adenosine kinase or cytosolic 5′-nucleotidase II, albeit with a much lower activity. The catalytic efficiency for viramidine phosphorylation is 10- to 330-fold lower than that of ribavirin, suggesting that other nucleoside kinase(s) may be involved in viramidine phosphorylation in vivo. Both ribavirin and viramidine are not phosphorylated by deoxycytidine kinase and uridine-cytidine kinase. The coincidence of presence of high concentrated 2,3-bisphosphoglycerate in erythrocytes suggests that cytosolic 5′-nucleotidase II could play an important role in phosphorylating ribavirin and contribute to anabolism of ribavirin triphosphate in erythrocytes. Elucidation of ribavirin and viramidine phosphorylation mechanism should shed light on their in vivo metabolism, especially the ribavirin-induced hemolytic anemia in erythrocytes.