Site-directed mutagenesis was used to replace asparagine (Asn) residues with glutamine (Gln) at the five potential N-linked glycosylation sites located at positions 28, 97, 138, 143, and 182 in the extracellular domain of the porcine growth hormone receptor (pGHR). These mutated pGHR cDNAs were stably expressed in mouse L cells. Single substitution of the Asn residues did not alter growth hormone binding when compared to cells which express native pGHR (KD approximately 1 nM). However, substitution of the five potential Asn-linked sites together (pGHR delta 5) resulted in a 20-fold reduced GH binding affinity (KD = 20 nM). Residues Asn97, Asn138, and Asn182 were apparently glycosylated and upon cross-linking with 125I-labeled pGH migrated as a molecular complex of approximately 130 kDa. Native pGHR and pGHR analogs with substitutions of N28Q and N143Q when cross-linked to 125I-labeled pGH, migrated with a Mr of 138 kDa. The fully deglycosylated cross-linked receptor, pGHR delta 5, migrated as a complex of 108 kDa. Therefore, each carbohydrate moiety contributed approximately 10 kDa to the total molecular mass of the pGHR, in sum contributing 30 kDa to the total Mr of the glycosylated pGHR. pGHR delta 5 was able to internalize nearly all the bound 125I-labeled pGH within 10 min, whereas native pGHR and individual Asn substituted pGHR analogs internalized 25% of bound 125I-labeled pGH at 10 min. Also, mutagenesis of the pGHR five potential Asn-linked glycosylation sites, either singly or together, did not alter the ability of GH to induce tyrosine phosphorylation of a 95-kDa protein. Together, the results indicate that three of the five pGHR Asn residues are apparently glycosylated and are necessary for maintenance of a high affinity GH binding site and for GH internalization. However, glycosylation of the pGHR is not critical for eliciting tyrosine phosphorylated proteins following the GH/GHR interaction.