Ove mutants in the moss Physcomitrella patens can arise from different recessive mutations. These mutants display a much larger number of buds than the wild type (wt) due to a dramatic overproduction of cytokinins (Cks), which are released into the culture medium (T.L. Wang, R. Horgan, D.J. Cove [1981] Plant Physiol 68: 735-738). The amounts of isopentenyladenine (iP) and isopentenyladenosine ([9R]iP) produced by chloronema of different ove mutants were measured. Levels of the major Ck iP in the culture medium of the mutants oveA78, oveA201, oveC200, and oveB300 (cultured at 21 degrees C) were 4-fold (oveA78) to 22-fold (oveB300) higher than for the wt. A new temperature-sensitive ove strain oveST25, which exhibits a strong ove phenotype at 25 degrees C, was also studied. It produced about 260 times more iP than the thiamine auxotrophic wt from which it was derived. To contribute to the physiological understanding of Ck overproduction, in vivo labeling experiments with (3)H-[9R]iP were performed. In all ove mutants analyzed, the rate of (3)H-[9R]iP conversion to (3)H-iP was higher as compared with the wt. In oveST25, the 3-fold increased riboside to base conversion was temperature inducible and correlated with the iP production. Analysis of Ck catabolism revealed no major differences between ove mutants and wt, thus indicating that ove mutants are unlikely to be degradation mutants. The data suggest that in ove mutants the increased riboside to base conversion is part of a generally up-regulated Ck biosynthetic pathway and may play an important role for the enhanced release of iP into the medium.