Two DNA probes representative of either the entire mouse mammary tumor virus (MMTV) genome or the poly(A)-adjacent sequences at the 3' end of MMTV RNA were synthesized with calf thymus DNA or oligo(dT) primers, respectively. These probes were used to study the expression of endogenous MMTV sequences in several BALB/c mammary tumor cell lines, in normal lactating BALB/c tissue, and in a cloned C3H tumor cell line. Both probes were characterized with respect to their rates of hybridization with template RNA, their size as determined by alkaline sucrose gradient centrifugation, and the thermal stability of the cDNA.MMTV RNA hybrids. In addition, the ability of the calf thymus oligodeoxy-nucleotide- or oligo(dT)-primed probes to protect (125)I-labeled MMTV RNA or (125)I-labeled poly(A)-adjacent MMTV RNA sequences from S1 nuclease digestion was determined. Hybridization analysis with these two probes indicated that (i) there were approximately 20-fold more oligo(dT)-primed sequences in BALB/c lactating tissue than there were sequences representing the entire genome; (ii) in BALB/c tumor cells, the oligo(dT):random oligonucleotide-primed cDNA sequence ratio was reduced to 4:1; and (iii) in virus-producer C3H tumor cells, there was only a 2-fold excess of oligo(dT)-primed sequences over that observed with a representative cDNA. These results are consistent with the presence of subgenomic viral mRNA species, integration of partial proviral copies, or altered mRNA processing.