Previously, we investigated courtship behaviour of bidirectional, interspecific hybrid males of two species of Nasonia (Beukeboom & van den Assem, 2001). Characteristics of the displays were intermediate between those of the parental species, but at the same time were biased towards the paternal side. Due to the haplodiploid sex determination system of the Hymenoptera, the nearest male parent of the haploid (hence, fatherless) Nasonia males are their grandfather. Therefore, we have called this bias the 'grandfather effect'. In the present paper, we investigate one of the possible causes of the 'grandfather effect': nucleocytoplasmic interaction. In interspecific hybrids, the paternally donated nuclear genes must operate in an 'alien' environment: the maternal (heterospecific) cytoplasm. Adverse effects may prevail in this situation, and result in a biased gene transmission (although to the maternal side). With introgression techniques and subsequent hybridisation of introgressed lines, we constructed male progeny in which paternally contributed nuclear genes are conspecific with the maternal cytoplasm. Courtship of these males provide a test of the interaction hypothesis. Because our results show a persistence of the 'grandfather effect', a simple nucleo-cytoplasmic interaction seems an unlikely explanation of the phenomenon.