The karyotype, chromosomal measurements, and the time course of DNA replication during the S-phase were determined in metaphase chromosomes of non-synchronized monolayer cultures of PtK2 cells (CCL 56) derived from Potorous tridactylis. The karotype was the same as originally determined for this cell line. Chromosomal measurements differed from data for primary bone marrow cells of this species published by Shaw and Krooth. PtK2 cells and chromosomes showed maximal incorporation of tritiated thymidine (3H-TdR) halfway through the S-phase. Chromosome Y1 showed a second peak of 3H-TdR-incorporation at the end of the S-phase in addition to the peak halfway through S. Comparison of grain densities for chromosomal arms showed late replication of the short arms of chromosomes 1, 3, and X. The time course of incorporation of 3H-TdR was changed when cells were treated for 1 h with fluorodeoxyuridine (FUdR) prior to the 3H-TdR-pulse. FUdR-treated cells showed maximum incorporation of 3H-TdR immediately after the beginning of the S-phase, which was followed by a second peak halfway through the S-phase. This indicated that 3H-TdR-incorporation was partially synchronized by treatment of cells with FUdR. Total radioactivity of FUdR-treated cells had increased by 77% in comparison to cells not treated with FUdR, which indicates that approximately 44% of the TdR-precursors of the latter cells may have originated from cellular precursor pools.