SEN virus (SEN-V) is a recently identified single-stranded, circular DNA virus. Two SEN-V variants (SENV-D and SENV-H) were assayed by polymerase chain reaction (PCR) to investigate their role in the causation of transfusion-associated non-A to E hepatitis. The incidence of SEN-V infection after transfusion was 30% (86 of 286) compared with 3% (3 of 97) among nontransfused controls (P < .001). Transfusion risk increased with the number of units transfused (P < .0001) and donor-recipient linkage for SEN-V was shown by sequence homology. The prevalence of SEN-V in 436 volunteer donors was 1.8%. Among patients with transfusion-associated non-A to E hepatitis, 11 of 12 (92%) were infected with SEN-V at the time of transfusion compared with 55 of 225 (24%) identically followed recipients who did not develop hepatitis (P < .001). No effect of SEN-V on the severity or persistence of coexistent hepatitis C virus (HCV) infection was observed. In 31 infected recipients, SEN-V persisted for greater than 1 year in 45% and for up to 12 years in 13%. SEN-V-specific RNA (a possible replicative intermediate) was recovered from liver tissue. In summary, SENV-D and -H were present in nearly 2% of US donors, and were unequivocally transmitted by transfusion and frequently persisted. The strong association of SEN-V with transfusion-associated non-A to E hepatitis compared with controls raises the possibility, but does not establish that SEN-V might be a causative agent of posttransfusion hepatitis. The vast majority of SEN-V-infected recipients did not develop hepatitis.