The oncogene v-myb of avian myeloblastosis virus is expressed from an mRNA that arises by splicing of the viral genome. In previous work, we described a mutant strain of avian myeloblastosis virus (tsAMV) that elicits temperature-sensitive transformation and suggested that the mutation affects production of the mRNA for v-myb. We now report that the principal determinant of the biochemical phenotype of tsAMV is a point mutation located in a crucial region of the splice acceptor site for v-myb mRNA. The mutation reduces v-myb mRNA production but could account for the conditional phenotype only in combination with an independent effect of temperature on the splicing of both wild-type and mutant viral RNAs, which we also describe here. Our findings dramatize the manner in which retroviruses normally control the splicing of their RNAs and implicate the sequence of the splice acceptor site in the control.