The in vivo synthesis of polycistronic transcripts of vesicular stomatitis virus in human amnion U cells and mouse L cells was detected by RNA blot hybridization. Within the molecular weight range resolved by this gel electrophoresis system, all possible combinations of sequentially linked messages were observed, as identified by their patterns of hybridization and their apparent molecular weights. Actinomycin D pretreatment of mouse L cells did not affect the frequency or size of polycistronic messages, nor did these differ between L cells and U cells. Vesicular stomatitis virus polycistronic transcripts were synthesized in vivo in a roughly uniform distribution, except for the NS-M dicistronic mRNA, which was much more frequent. Most of the polycistronic RNA species were found to be poly(A)+, but at least one, the tetracistronic molecule N-NS-M-G, was clearly poly(A)-. Analysis of RNA following treatment with RNase H in the presence of oligo(dT) indicated that the in vivo-synthesized poly(A)+ polycistronic species NS-M, M-G, and N-NS-M had poly(A) tracts at their 3' molecular termini but not internally at their intercistronic junctions.