The morphological stability of hairy root cultures of Pimpinella anisum was studied using cultures grown in four different media, both in darkness and under photoperiod conditions of 16 h light; they were first subcultured every eight days and then later on every three weeks. From the four media tested, the hairy root cultures grown in SH medium both in darkness and under photoperiod conditions showed a marked morphological stability with no de-differentiation or greening, when compared with the other culture systems. The time-course study of the essential oils, isolated by distillation-extraction and analysed by GC and GC-MS, showed only quantitative differences in the composition of the oils. A clear heterogeneity in the accumulation pattern was found with regard to the five dominant compounds (pregeijerene, geijerene, zingiberene, beta-bisabolene and trans-epoxypseudoisoeugenyl 2-methylbutyrate), i.e., those that appeared in a relative amount higher than 5% at least once during the time-course study of the oils from the eight culture systems.