Prostaglandin production, angiotensin-converting enzyme, and 5'-nucleotidase were measured in porcine aortic endothelial cells in situ (with a multiwell template on an opened aorta), in primary culture and in subcultures. Changes during culture were monitored and the effects of culture conditions were investigated by growing cells on a biological matrix or on plastic, by adding different sera to the growth medium, and by harvesting cells enzymically or mechanically. Prostacyclin production by endothelium in primary culture is highest immediately after cell isolation and subsequently declines; this pattern is repeated each time the cells are subcultured. The level at which production stabilises is -200 pg.106 cells-'. h -I . Detaching cells by physical means stimulates production much more than enzymic dispersion; the type of serum or the presence of a biological matrix does not alter prostaglandin production. The relative amount of prostaglandin E produced increases with time, from -20% of the prostacyclin production shortly after isolation to >loo% in subcultured cells. None of the culture conditions that we tested altered this trend. Angiotensin-converting enzyme activity decreases during primary culture, but activity can be sustained by including homologous serum (from whole blood or from platelet-free plasma) in the culture medium. The method of harvesting cells, or the presence of a matrix, did not affect enzyme activity. 5'-Nucleotidase also declines during culture, with a progressive decrease in both K, and V,,, from template to primary culture to subcultures. None of the variations in culture conditions prevented this change. Ectoadenosine-deaminase activity, not detectable in cultured cells, can be measured in the template. Part of this activity was released by the vascular wall and could be due to plasma diffusing from the interstitial space.