This study was carried out to better understand factors that influenced the process of attenuation of Marek's disease (MD) virus by serial passage in cell cultures. Three virulent (v) pathotype and three very virulent plus (vv ) pathotype strains were passed by three techniques up to 131 times, and the passage level at attenuation was determined. The 18 attenuated or partially attenuated viruses were evaluated for protection against challenge with virulent MD virus, and the virus load (latent infection) in blood lymphocytes at 14–21 days postvaccination was determined. Viral pathotype strongly influenced the rate of attenuation. The mean passage level at attenuation for v and vv strains was 74 and >109, respectively. Full attenuation was achieved for nine of nine passage series with v pathotype strains but for only four of nine passage series with vv pathotype strains. Time to attenuation was not significantly influenced by multiplicity of infection at passage or by cell type, although a possible advantage of alternate high- and low-multiplicity passage was noted. Protection was not significantly influenced by pathotype or time to attenuation. Protection varied from 50% to 95% for the 18 passaged virus preparations; six attenuated viruses provided high protection that did not differ from that of the prototype Rispens strain. Virus load was not influenced by pathotype or by passage strategy and showed no positive correlation with protection. In several Cases the most protective vaccines had the least virus load. This finding differs from previous reports and warrants further study. Variation among different strains within the same pathotype was documented for attenuation rate, protection, and virus load. Also, variation was evident when the same strain was passaged by different strategies, probably reflecting random changes during serial passage. Strain 596A (v pathotype) was the first to become attenuated, provided the best protection, and had one of the lowest virus loads. In contrast, strain 617A (v pathotype) provided the least protection and had one of the highest virus loads. Such strains provide fertile opportunities for further study.