Small amounts of fractionated, denatured, (32)P-labeled DNA from SV40 virus were incubated with a large excess of the complementary RNA of SV40 prepared in vitro with Escherichia coli RNA polymerase; the viral DNA strands were separated on hydroxyapatite columns. The RNA present in green monkey cells late in the lytic cycle reacted with 40-42% of the strand complementary to the in vitro complementary RNA (minus strand), and 60-64% of the opposite (plus) strand. "Early lytic" RNA failed to significantly interact with the plus strand, but formed stable duplex molecules with 35-39% of the minus strand. The RNA prepared from mouse embryo cells 24 hr after infection with SV40 combined with 35-38% of the minus strand and 60-62% of the plus strand. In all cases, the same regions of either the plus or minus strand appear to be transcribed in permissive and nonpermissive infections.