A high frequency (.65%) of thymomas induced by mink cell focus-forming virus 69L1 in AKR/J mice contain proviral integrations which are clustered 0.7-kilobase upstream of the c-myc oncogene predominantly in the opposite transcriptional orientation. Blot hybridization experiments showed that on the average there was only a twofold elevation of steady-state c-myc RNA in the thymomas as compared with levels in normal AKR/J thymocytes. Such an increase would not appear to be sufficient as a mechanism of oncogene activation in this system. In contrast, Si nuclease analysis of transcripts initiated from the two known c-myc promoters indicated a strong shift in promoter usage in virtually all thymomas tested. In normal thymus the ratio of transcripts initiated from the proximal promoter P1 to the distal promoter P2 was 0.2 to 0.3. In contrast, most of the thymomas tested (18 of 23) showed an average P1/P2 ratio of 1.2 regardless of whether or not proviral integrations could be detected within a 21-kilobase EcoRI fragment containing the three c-myc exons. We conclude that alterations in P1/P2 ratios are good indicators of c-myc deregulation in thymic lymphomas.The c-myc oncogene has been implicated in a variety of malignant neoplasms. In lymphoid tumors of both B-and T-cell lineages activation of c-myc has been associated with DNA rearrangements at the c-myc locus caused by either chromosomal translocations or retrovirus insertions (for-a recent review, see reference 25). Recently, we reported murine leukemia virus proviral integrations into the c-myc locus in 65% of thymic lymphomas induced by intrathymic injection of mink cell focus-forming virus (MCF) 69L1 into 40-day-old AKR/J mice (13). In this report we measured steady-state levels of both total c-myc RNA and c-myc transcripts initiated from each of the two known promoters of the gene to study the effect of proviral integration on expression of the c-myc oncogene. We present evidence that expression of c-myc is not increased markedly in MCFinduced thymomas but is deregulated in tumors as evidenced by a reversal of normal promoter usage. Altered levels of the two c-myc transcripts also were observed in many instances in which a proviral integration was not detected near c-myc.A map of MCF proviral integration sites for 40 independent primary thymomas which have been analyzed to date is shown in Fig. 1. Most (32 of 40) of the proviral integrations were in the reverse transcriptional orientation to c-myc and were distributed about a median distance 0.7-kilobase (kb) 5' of exon 1, a finding similar to that reported by other workers (3,9,19). Of the eight tumors with colinear proviral integrations, only two of them, with insertions at +0.7 kb and +5.2 kb, were available for analysis in this study.Steady-state levels of c-myc RNA were measured by slot-blot analysis of total cellular RNA (Fig. 2a). Samples of total RNA (2) in 4.6 M formaldehyde-7.5 x SSC (0.1 M sodium chloride, 0.01 M sodium citrate) were heated at 65°C for 15 min, diluted serially, and applied to two se...