Astrocyte proliferation and differentiation in primary culture were fo
llowed by flow cytometry, The time-courses of the percentages of astro
cytes in the different cell-cycle phases suggest that astrocytes proli
ferate during the first 10 days in culture thereafter reaching conflue
nce. Two types of astrocytes are identified immunocytochemically: one
growing in the bed monolayer, identified as type-1 astrocytes, and ano
ther growing on the top of the monolayer, identified as type-2 astrocy
tes. In addition, three populations identified as being formed of type
-1, type-2 and putative progenitor cells, respectively, were followed
by flow cytometry. Progenitor cells were the major type 2 h after plat
ing (89%) but their percentage decreases sharply (to 16%) during the f
irst 5 days in culture, with no ensuing changes. In contrast, the perc
entage of type-1 cells (11%) rapidly increased after plating reaching
a maximum 5 days later (73%). Later, it decreased (to 47%) and was mai
ntained thereafter. The percentage of type-2 cells was undetectable im
mediately after plating but increased from the 3rd to the 10th day wit
h no further changes. Our results suggest that progenitor cells differ
entiate into type-1 astrocytes triggered by the culture medium but the
differentiation of progenitor cells into type-2 astrocytes is brought
about by some type-1-secreted factor. In this work we report a rapid
and simple method for following the growth and differentiation of rat
brain astrocytes in primary culture.