A real-time PCR was used to measure increases in viral DNA in Marek's disease virus (MDV)-infected primary chicken cell cultures in order to optimize methods for viral isolation. Serotype-1 and -3 vaccine and serotype-1 challenge strains exhibited similar growth characteristics, with increases in viral DNA being proportional to inoculum size. Studies of viral growth revealed a linear relationship between increase in MDV copy number and infectious titre, although the rate of increase for copy number was greater. Using real-time PCR, viral DNA yields of the virulent Woodlands strain in infected chicken kidney cultures were shown to be slightly, but not significantly, higher than in chicken embryo kidney cultures and significantly higher than in chicken embryo fibroblast cultures. Viral DNA levels in freshly trypsinised cells suspended in growth medium and infected with the Woodlands strain were higher than levels obtained following the inoculation of monolayer cultures. For cells infected in suspension, no significant enhancement of yield was observed following a medium change after 2-3 days. Peak yields were obtained at days 6-8 after inoculation of all cultures. Findings obtained from the optimization of viral DNA levels were applied to a program for the isolation of Australian strains of serotype-1 viruses from problem flocks over 3 years. Significant improvements were obtained in the isolation rate of strains capable of growing to high titre (>10(4) plaque-forming units/mL) for use in challenge studies.