Kasanosins A (1) and B (2) are novel azaphilones isolated from cultures of Talaromyces sp. derived from seaweed, and their structures were determined by spectroscopic analyses. These compounds selectively inhibited the activities of eukaryotic DNA polymerases beta and lambda (pols beta and lambda) in family X of pols, and compound 1 was a stronger inhibitor than compound 2. The IC(50) values of compound 1 on rat pol beta and human pol lambda were 27.3 and 35.0 microM, respectively. On the other hand, compounds 1 and 2 did not influence the activities of terminal deoxynucleotidyl transferase (TdT), which is a pol of family X, and the other families of eukaryotic pols, such as family A (i.e., pol gamma), family B (i.e., pols alpha, delta, and epsilon) and family Y (i.e., pols eta, iota, and kappa), and showed no effect even on the activities of plant pol alpha, fish pol delta, prokaryotic pols, and other DNA metabolic enzymes, such as calf primase of pol alpha, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, mouse inosine 5'-monophosphate (IMP) dehydrogenase (type II), human topoisomerases I and II, T4 polynucleotide kinase, and bovine deoxyribonuclease I. The results suggested that these novel compounds could identify the inhibition between pols beta, lambda, and TdT in family X.