The substrate specificity of choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from brewer's yeast has been examined using multiple analogs of choline, most of which have been reported to be a substrate of one or another choline-using system from other sources. In contrast to many such systems, choline kinase from brewer's yeast has been found to have relatively stringent and straight-forward structural requirements for its substrates. It is hypothesized that there are at least four points of interaction of the substrate with the enzyme--one for the hydroxyalkyl side chain and one for each of the three substituents on the quaternary nitrogen. Of the latter, one site seems relatively more sterically hindered than the other two. Short, single or double alkyl substitutions on the quaternary nitrogen are possible without a large loss of substrate capacity of the analog. Thus N,N-dimethyl-N-propylethanolamine had a relative Vmax of 116% and a relative Vmax 96% that of choline and a Km of 68 +/- 15 microM [nearly four times that of choline itself (18 microM)]. However, N-butyl-N,N-dimethylethanolamine and N,N,N-triethylethanolamine were very poor substrates. Analogs with substituents on the quaternary nitrogen of longer chain length were without activity as were aromatic derivatives. None of the bisquaternary compounds of the general structure HOCH2CH2N+(CH3)2-(CH2)n-N+(CH3)2CH2CH2OH (n = 2-10) showed any substrate capacity, as well. Restrictions on the hydroxyethyl side chain were also severe. One additional methylene group in this chain greatly reduced substrate capacity of the analog and two additional ones eliminated it entirely, as did almost any substituent on the beta carbon. A single (but not a double) substituent on the alpha carbon was moderately tolerated, however. Thus alpha-methylcholine and N-methyl-2-hydroxymethylpiperidine were substrates (although the latter one was a poor one) but beta-methylcholine and N-methyl-3-hydroxypiperidine were not. Such information may be of use toward designing cholinergic probes targeting specific enzyme or metabolic functions.