Penelope-like elements have been described in recent years in various animals from rotifers to fish and reptiles. In our previous studies, the injection of Penelope-containing constructs into the embryos of a D. virilis strain 9 lacking active Penelope resulted in multiple mutations in the progeny. It was shown that almost half of all visible mutations isolated in these experiments were due to insertions of Ulysses, which, contrary to Penelope, has nearly symmetrical distribution in the parental strains. Recently, we have monitored the biogenesis of small RNAs homologous to various D. virilis transposons and measured the transmission levels of corresponding siRNAs and piRNAs in various inter-strain crosses. Using P-like strain 160 and a few neutral D. virilis strains that contain multiple full-size and potentially functional Penelope copies, however, we detected no obvious correlation between dysgenic traits and maternally deposited Penelope-derived piRNA levels. Therefore, we sought to expand these studies in order to reveal correlations between the levels of naturally occurring transposition in D. virilis WY 14643 50892-23-4 laboratory strains and RNA production and/or the biogenesis of the TE-derived small RNAs in question. Herein, we demonstrate asymmetric transposition of Penelope and Ulysses in the laboratory strains of D. virilis without performing dysgenic crosses. By RNA whole-mount in situ hybridization a different subcellular strain specific localization of the TEs transcripts was revealed. Furthermore, we show that processing of Penelope and Ulysses transcripts lead to the formation of different classes of small RNAs that may be implicated in transposition control of these TEs. For comparison, we have also investigated expression of gypsyDv, which is based upon previous studies lost transposition activity in D. virilis and is not mobilized by dysgenic crosses in this species. While we did not find new sites for Ulysses in strain 160, we did reveal active transposition of this TE in M-like strain 9. It is noteworthy that all the chromosomes of strain 9 were involved in the transposition process by Ulysses. It is necessary to note that even though transpositions of retroelements do not occur by a “cut and paste�?mechanism, in strain 9 we detected six new sites of insertion in parallel with the disappearance of four “old�?sites detected in 1991. Such a phenomenon was described in D. melanogaster, when certain copies of the retroelement gypsy or Ielement disappeared without a trace from a few cytological locations. Characteristically, the presumably inactive gypsyDv taken for comparison exhibited practically identical preferentially heterochromatic distribution in the chromosomes of the D. virilis strains studied, which was preserved without any change during the whole period of observation.