Virions from Newcastle disease virus mutants in four temperature-sensitive RNA+ groups were grown in embryonated hen eggs at the permissive temperature, purified, and then analyzed for biological properties at both the permissive and nonpermissive temperatures. At the permissive temperature, virions of mutants in groups B, C, and BC (11 mutants) were all lower in specific (per milligram of protein) hemagglutination, neuraminidase, and hemolysis activities compared with the wild type. These deficiencies were related to decreased amounts of hemagglutinin-neuraminidase glycoprotein in the virions. Activities of these mutant virions at both the permissive and nonpermissive temperatures were similar, indicating that hemagglutinin-neuraminidase synthesized at the permissive temperature was not temperature sensitive in function. The three group D mutants displayed a different pattern. At the permissive temperature, they had wild-type hemagglutination and neuraminidase activities but were deficient compared with the wild type in hemolysis. Again, functions were similar at both temperatures. Most of the B, C, and BC mutants had specific infectivities similar to that of the wild type despite lower hemagglutination, neuraminidase, and hemolysis functions. However, the D mutants were all less infectious. This evidence is consistent with a shared hemagglutinin-neuraminidase defect in the B, C, and BC mutants and a defect in either the F glycoprotein or the M protein in the D mutants.