The influence of several factors on parasite growth and 50% inhibitory concentration (IC(50)) for chloroquine was assessed. Most isolates stored at 4 degrees C up to 72 hours grew when they were subsequently cultivated. However, parasite viability sharply decreased from 24 hours, and the mean chloroquine IC(50) decreased significantly (P < 0.05). There was no evidence for selection of pre-culture populations due to storage alone. The time point when (3)H-hypoxanthine was added (0 versus 18 hours) had no effect on the IC(50) during the 42-hour incubation, but was associated with a lower IC(50) when (3)H-hypoxanthine was added after the initial 42-hour incubation during the 72-hour incubation. An increase in 3H-hypoxanthine incorporation and chloroquine IC(50) was observed as the hematocrit was increased from 1.0% to 2.5%. For the same isolates, chloroquine IC(50) values were generally similar when the initial parasitemia was between 0.1% and 0.5% but increased at higher (>0.75%) parasitemias. Based on these results, we recommend immediate cultivation after blood collection, a 42-hour incubation period with the addition of (3)H-hypoxanthine at the beginning of incubation, a 1.5% hematocrit, and an initial parasitemia 0.1-0.5%. Further studies on serum substitutes, gas mixture, and comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol.