Primary cultures of rat myocardial capillary endothelial cells were established and characterized. A range of typical endothelial cell-specific markers were retained in vitro. Cell kinetic studies in confluent endothelial-cell cultures in vitro revealed a roughly 50-fold increase in the proportion of cells in s-phase, indicating a very considerable shortening of cell turnover time, compared to in vivo conditions. Alkaline phosphatase enzyme activity and encoding mRNA are strongly expressed in myocardial capillary endothelial cells in vivo, but were not detectable in vitro. This was true in cell cultures from two strains of rat, which revealed significantly different enzyme expression levels in vivo. In co-cultures of pericytes and endothelial cells, positive ALP enzyme reaction was detected in pericytes, which in vivo show only very weak enzyme reactivity. Treatment of cell cultures with