While a number of osteotropic hormones regulate insulin-like growth factor-I (IGF-I) synthesis in osteoblast-enriched (Ob) and intact bone cultures, their direct effects on IGF-II production are still unresolved. For example, cAMP stimulators, such as PTH and prostaglandin E2, increase Ob IGF-I transcript and polypeptide levels within the first 24 h of treatment, but have no effect on IGF-II expression. To examine the possibility that other circulating factors could directly modify IGF-II synthesis by osteoblasts, primary rat Ob cultures were briefly treated with a number of polypeptide and steroid hormones known to regulate bone metabolism. Prepro-IGF-II steady state transcripts were assessed by Northern blot analysis, and immunoreactive polypeptide levels (iIGF-II) were examined by RIA. Predominant prepro-IGF-II transcripts of 3.7 kilobases were readily detected in quiescent Ob cultures, and constitutive iIGF-II levels were approximately 2-7 nM throughout the first 24 h of culture. GH, placental lactogen, insulin, cortisol, testosterone, T3, 17 beta-estradiol, and 1,25-dihydroxyvitamin D3 each had no effect on prepro-IGF-II transcripts within 6 h or on iIGF-II polypeptide expression within a 24-h period. These studies indicate that IGF-II synthesis is constitutive in unstimulated primary fetal rat Ob cultures, and that these levels are not directly modulated by short term treatment with a variety of osteotropic hormones.