When grafting human mesencephalic tissue to patients suffering from Parkinson's disease, the number of surviving dopamine (DA) neurons in the graft is probably crucial. It may be possible to increase the number of DA neurons available for grafting to a patient by pooling tissue from many human embryos collected over several days or by obtaining more DA neurons from each embryo. We have addressed these issues by cryopreserving human mesencephalic DA neurons prior to transplantation and also by grafting human embryonic diencephalic DA neurons. The effects of cryopreservation were assessed 4-15 weeks after xenografting ventral mesencephalic tissue into the DA-depleted striatum of immunosuppressed rats with unilateral 6-hydroxydopamine lesions of the mesostriatal pathway. Control rats grafted with fresh mesencephalic tissue displayed robust reductions in amphetamine-induced turning following transplantation. Functional effects of the cryopreserved mesencephalic grafts were only observed in the one rat out of nine which contained the largest graft in this group. The number of tyrosine hydroxylase immunoreactive neurons in animals transplanted with cryopreserved tissue was significantly reduced to 9% of fresh tissue control grafts. Morphological analysis showed that cryopreserved DA neurons were approximately 22% and 28% smaller regarding the length of the long and short axis, respectively, when compared to the neurons found in fresh grafts. In the second part of the study, the survival and function of human embryonic diencephalic DA neurons were examined following xenografting into the DA-depleted rat striatum. A reduction of motor asymmetry was observed in two out of seven diencephalon-grafted rats. This finding was consistent with a good graft survival in these particular rats, which both contained large grafts rich in tyrosine hydroxylase immunoreactive neurons. Moreover, there was immunopositive staining for graft-derived fibers in the rat striatum containing tyrosine hydroxylase and human neurofilament, both in rats grafted with mesencephalic and diencephalic DA neurons. These findings suggest that cryopreservation, using the current technique, is not a suitable storage method for use in clinical trials of DA neuron grafting in Parkinson's disease. On the other hand, the application of alternative sources of DA neurons may in the future develop into a strategy which can increase the number of neurons obtainable from each human embryo.