Ribokinase (EC 22.214.171.124) from Leishmania major was cloned, sequenced and overexpressed in Escherichia coli. The gene expressed an active enzyme that had comparable activity to the same enzyme studied in E. coli. It specifically phosphorylated D-ribose. Under defined conditions, the K(m) for the substrates D-ribose and ATP were 0.3+/-0.04 mM and 0.2+/-0.02 mM, respectively. The turnover numbers of the enzyme for the substrates were 10.8 s(-1) and 10.2 s(-1), respectively. The enzyme product ribose 5-phosphate inhibited the phosphorylation of D-ribose with an apparent K(i) of 0.4 mM, which is close to the K(m) (0.3 mM) of D-ribose, suggesting that it might play a role in regulating flux through the enzyme.