To provide information on the mechanism of attenuation of a Newcastle disease vaccine strain, TCND, we compared it with the parental virulent strain California 11,914 (CAL) biologically and genetically. It was found that TCND bore the fusion protein of virulent type, consisting of a pair of dibasic amino acid residues at the cleavage site and was a temperature sensitive (ts) mutant restricted to grow at 41.5 degrees C. Revertants were obtained by prolonged incubation of chicken embryos inoculated with TCND at the nonpermissive temperature. In cultured cells, viral gene transcription and protein synthesis of TCND occurred similarly to those of CAL and the revertants at 41.5 degrees C. Hemadsorption and immunofluorescence assays revealed that cell surface expression of functional hemagglutinin-neuraminidase (HN) of TCND at 41.5 degrees C was lower than that at 35 degrees C. The revertants exhibited lower activity in fusion assay than CAL and recovered virulence to chicken only in part. The results indicate that the ts mutation of TCND in association with the defect of HN glycoprotein transport is a mechanism of the attenuation, and in addition, some other factors such as fusion activity should be involved in the loss of virulence of CAL to chickens.