Regulation of the production and secretion of sex hormone-binding globulin (SHBG) was investigated in HepG2 cell cultures by measuring SHBG protein concentrations intra- and extracellularly and studying changes in SHBG messenger ribonucleic acid levels. Insulin (10 nmol/L), insulin-like growth factor-I (15 nmol/L), and epidermal growth factor (20 nmol/L) decreased SHBG levels in parallel both intra- and extracellularly. Ten nmol/L 17 beta-estradiol, 10 nmol/L testosterone, and 100 nmol/L to 1 mumol/L cortisol increased SHBG levels inside the cells, but did not increase its release into the culture medium. Two hundred and fifty to 500 nmol/L 17 beta-estradiol and 500 nmol/L to 1 mumol/L testosterone increased SHBG levels intra- and extracelularly, but relative to control values, the increase was considerably greater inside the cells. T3 (10 nmol/L) increased SHBG levels, but unlike the effect seen with steroids, the increase was equally evident within the cells and the medium. Northern hybridization showed that insulin decreased and 17 beta-estradiol and T3 increased SHBG messenger ribonucleic acid levels marginally. The variable secretion of SHBG is hypothesized to be due to the different effects of hormones and growth factors on either the glycan moiety of SHBG or the expression of the alternatively spliced transcripts of the SHBG gene.