Yeast cytosine deaminase (yCD) was cloned, expressed, and purified by affinity chromatography. We have characterized the products resulting from covalent attachment of 2-4 PEG chains on yCD and determined the major and minor isomers for each respective conjugate. The results show that for non-covalently associated homodimers, it is possible to characterize and deduce PEGylation levels on individual subunits through the concurrent use of size exclusion chromatography (SEC), MALDI-TOF MS, and SDS-PAGE gels. The results also show that contrary to what we expected, attaching more than two PEG chains to yCD decreased its stability. Enzymatic activity studies revealed that the fusion of an N-terminus purification tag on yCD has no significant effect on 5-fluorocytosine or cytosine affinity, with apparent turnover rates remaining within 10(5) M(-1) . s(-1). Stability studies at 37 degrees C revealed that t1/2 = 8-9 h for yCD and 2mPEG(5K)-yCD, whereas for 3-, 4mPEG(5K)-yCD and yCD/BSA, t(1/2) < 2 h. Incubation of BSA with yCD also decreased enzyme stability over prolonged incubation at 37 degrees C. This finding is important if yCD is to be used in a pretargeting strategy.